For bioenergy research, CAZymes are obviously the most important enzymes. The CAZyDB team started to classify and annotate CAZyme proteins from Gen — Bank, UniProt and PDB to protein families since the early 1990s. It is the original database that defined over 300 CAZyme protein families throughout the past 20 years and the most comprehensive database providing high-quality manual annotation by extracting associated knowledge from the literature (Cantarel et al.,

2009) . Its Web site regularly updates every few weeks, mainly by assigning new proteins in public databases to existing CAZyme families by sequence similarity search or creating new families if there are new bio­chemically characterized CAZyme proteins (supported by published papers) that do not belong to existing CAZyme families. Sometimes the functional annotation information (e. g. known activities) for some families is also updated if relevant literature came out.

Currently the database comprises five classes of pro­tein families: in addition to GHs and GTs, there are three other classes, carbohydrate esterases (CEs), polysaccha­ride lyases (PLs) and carbohydrate binding modules (CBMs). As aforementioned, GTs are used for building polysaccharides or glycolconjugates, while GH for breaking them. CE and PL are also used for breaking car­bohydrate molecules while using different mechanisms or cutting different chemical bonds. CBMs, as indicated by names, are structural modules used for recognizing and binding different sugars. Currently the five classes contain 94 GT families, 131 GH families, 16 CE families, 22 PL families and 64 CBM families. Each class also has an unclassified family, meaning proteins are annotated to belong to a certain class but are not able to be assigned to any existing families in that class. Each family is named with the class name followed by a sequential number, e. g. GT2. Note such name does not indicate any biochemical activity of each family. The reason is that these families are defined solely based on sequence similarity: there are many cases that one family contains

proteins characterized with different biochemical activ­ities. Recent efforts from the CAZyDB team suggest that further classification of family into subfamilies could be useful as subfamily may contain proteins with the same activity (Stam et al., 2006; Lombard et al., 2010; Aspeborg et al., 2012).

CAZyDB’s annotation also evolved in the past 20 years. Among the 327 CAZyme families as of December 2012, there are 10 depreciated families; they were created during the life course of CAZyDB but later were deleted since they were shown not related to carbo­hydrate metabolism or due to some other reasons. How­ever, to keep the existing nomenclature system unchanged, these family names remain in the system but indicated to be deleted on the Web pages for these families. Other examples include CE10 family, whose

Web page was not updated since 2002 because after the family was created it was shown that most CE10 family members do not take carbohydrates as substrate; CBM33 was thought to be a carbohydrate active binding module but later shown likely to be an oxidase family.

For a decade, CAZyDB provides an HTML page for each family to list member proteins and associated func­tional information. In recent updates, CAZyDB added a Web page for each genome, providing a list of GenBank protein accession numbers of that genome together with the CAZyme family assignment for each protein, which is termed "CAZyome" of an organism. So far, there are almost 2400 genomes spanning from eukaryotes to prokaryotes and viruses annotated in CAZyDB. It is said that such genome-scale annotation of CAZyme proteins is done semiautomatically (Coutinho and

Henrissat, 2011). A backend automated domain module — based search is performed first and then manual curation will be conducted to remove false positives or include false negatives. Obviously this process is rather accurate and of high quality but time consuming because it is done manually and requires expert knowledge. Indeed such genome annotation can only be done by the collab­oration with the CAZyDB team, which is often invisible to and out of the control of the users, e. g. people who sequenced the genome. Over the past 10 years, the CAZyDB team has done expert CAZyme annotation for dozens of genome sequencing projects that led to a lot of collaborative genome annotation papers.

To maximize biomass production and the accumula­tion of fuel precursors, algal cultures must be supplied with various concentrations of macronutrients, vita­mins, and trace elements depending on species require­ments. While there are limited reports on optimal levels of nutrients required for mass algal cultures, it is gener­ally accepted that required macronutrients are nitrogen and phosphorus (Brzezinski, 1985; Harrison and Berges,

2005) . Trace elements such as cobalt, copper, molybde­num, zinc, and nickel are likewise necessary, and in some species are considered to be effective in hydrogen production (Ramachandran and Mitsui, 1984). There ap­pears to be no consensus on the optimal ratios for these nutrients, even for specific species grown successively in the same system. Therefore, nutrients are often added in excess to avoid nutrient limitations (Richmond, 1999; Sanchez et al., 1999; Acien Fernandez et al., 2001).

One strategy to reduce costs associated with adding excess nutrients involves culturing microalgae in reclaimed water or wastewater blends. The use of algae to absorb nutrients in the wastewater processing stream has been widely employed by water treatment facilities (Megharaj et al., 1992; Tredici et al., 1992; Nurdogan and Oswald, 1995; Kaya and Picard, 1995; Craggs et al., 1995). The green microalga Scenedesmus obliquus has demonstrated vitality in urban wastewaters, registering growth rates similar to those reported for a complete syn­thetic medium. This freshwater alga tolerates a wide range of temperature and pH, making it versatile for water puri­fication (Kessler, 1991). Similar findings for other algal spe­cies continue to emerge, along with the energy return on investment analyses that confirm the utility of coupling scaled algal (EROI) production with nutrient reclamation from waste streams, resulting in decreased costs for both algal growth and water treatment (Beal, 2012b).

CONTAMINATION

Another barrier to the large-scale production of algae biofuels is the maintenance of axenic or nearly axenic cul­tures. In particular, cultivation systems that are open to the environment (e. g. open ponds) are easily susceptible to contamination by unwanted species if extreme care is not taken. A new open pond is typically inoculated with the desired strain of microalgae with the hope that the algae will aggressively proliferate and dominate the pond flora. Over time, it is likely that undesired species will be introduced, which may graze on the algae or outcompete the inoculated species and lead to severely reduced yields. Once a competitor has taken residence in a pond, it is extremely difficult to eradicate (Schenk et al., 2008). It is therefore crucial to aggressively monitor cultures to identify and eradicate contaminates as soon as possible. A number of strategies have been employed to minimize culture contaminations. Cultivating algal extremophiles that tolerate and outcompete invasive spe­cies in particular environments (e. g. pH and salinity) fa­cilitates open-pond production. High bicarbonate concentrations allow Spirulina to be grown in open ponds with few invasive algae, and high-saline environments allow marine algae like Dunaliella salina to be grown in "relative pure cultures" (Anderson, 2005). Another popu­lar strategy involves shortening the longevity of the cul­ture; cultures are scaled and harvested before major contamination can occur (Benemann, 2008). Cultivation of microalgae in closed photobioreactors (PBRs) offers another level of protection against predators. Occasion­ally, cultures can be treated with antibiotics and antifun — gals to eliminate bacteria and fungi, but this practice can lead to microbial resistance and render the treatment ineffective. Predator ciliates can be treated with dioctyl sulfosuccinate, which is used to eliminate ciliates in the udders of milking cows (Abou Akkada, 1968) with mini­mal harm done to the algae.

MIXING

At high algae concentrations, a thin top layer of cells absorbs nearly all light—this phenomenon can be avoided by proper mixing. Mixing must sufficiently keep algae cells in suspension, aid distribution of CO2 and O2, and provide uniform exposure of light to all cells. Mixing also decreases the boundary layer around cells, which facilitates increased uptake of metabolic products (Molina Grima et al., 1999).

As mentioned previously, the presence of FFAs in the biolipid is detrimental to oil quality and function, including biodiesel production. Removal typically in­volves the reaction of these FFAs with an alkaline solu­tion. In the edible oil industry, usually only caustic soda is used for this reaction, but potassium hydroxide is also used by some producers. The acidity of the FFA comes from the H+ of the carboxyl group. This H+ of the functional group of the stearic acid reacts with the OH~ group of the caustic soda (NaOH) to produce soap and water. In addition to the removal of FFAs, other undesirable nonglyceride materials are also removed in this fashion such as phenol, oxidized fatty compounds, heavy metals and phospholipids.

Winterization

Most biolipids do not need dewaxing, as they contain little or no waxes. Only biolipids of higher melting temperatures, such as sunflower oil and rice bran oil, give a hazy appearance during winter season due to precipitation of dissolved waxes. Hence, they require being dewaxed. This is carried out by chilling the oil to 10—15 °C, followed by filtration of precipitated solids. The oil thus treated has a sparkling appearance, even in winter temperatures.

Apart from clay materials, different types of layered compounds have been tested as heterogeneous catalysts in processes traditionally mediated by homogeneous catalysts, which are in some cases expensive and highly toxic. Reactions such as Michael addition, cyanoethyl — ations of alcohols, aldolic condensations and condensa­tion of nitro compounds with aldehydes and ketones, and ring openings can be used as examples (Centi and Perathoner, 2008).

Layered materials have also been used as solid cata­lysts for biodiesel production through esterification and transesterification. Most applications involving this class of compounds refer to the use of layered dou­ble oxides (LDOs), which are derived from layered dou­ble hydroxides (LDHs) by controlled calcination. LDHs, layered hydroxide salts (LHSs) and layered carboxylates are less commonly used for this purpose. This section presents a brief review of the structure of these layered materials in addition to the description of their use and performance as catalysts.

Layered Double Hydroxides

LDHs are compounds whose individual layers are of brucite-like (Mg(OH)2) structure. In brucite, the layers are electrically neutral with magnesium cations located in the center of an octahedron with six hydroxyl groups in the vertices. The isomorphic substitution of magnesium by trivalent cations forms positively charged layers, which are stabilized by the presence of anions in the interlayer space (Bravo-Suarez et al., 2004). LDHs are represented by the formula [M^M^+tpH^JA!^ nH20, where M2+ and M3+ are divalent and trivalent cations and Am~ represents an anion with an m-charge and x usually has a value between 0.25 and 0.33 (Crepaldi and Valim, 1998).

In this work, the chemical composition of a specific LDH will be condensed as M2+/M3+-A. Thus, an LDH whose layers contain Mg and Al and the counterion between the layers is nitrate will be written as Mg/ Al-N03.

0ne peculiar characteristic of LDHs is the memory ef­fect. Calcination of Mg/Al or Zn/Al LDHs forms mixed and nanostructured mixed metal oxides described as LD0, which are able to reassemble the LDH structure if added back to an aqueous solution containing a salt whose anions will be intercalated between the layers in order to stabilize the LDH structure (Carlino, 1997). These anions can be different from the anions found in the original LDH. This kind of materials can substitute basic homogeneous catalysts like ammonia, ammonium salts or amines and offer an option as nonpollutant solid catalysts that can be easily separated from the reaction system and recovered. Their catalytic activity is related to the large surface area of LDHs, its solid base character and layered morphology. For instance, Zn/Al LDH con­taining nitrate, sulfate or orthophosphate anions have catalytic activity in esterification reactions (Hu and Li, 2004), while Mg/Al LDH intercalated with t-butoxide is active in transesterification (Choudary et al., 2000).

Although the research activities show that in 2013 there is a great interest in using lignin as a renewable resource for the production of aromatic chemicals, it is also clear that commercial utilization will take substantial time. So far the literature results show that relatively low conversion yields to about 10 wt% based on dry lignin and resulting complex mixtures hinder the commercial utilization of these processes.

The Netherlands can play an important role in the lignin aromatics valorization technologies as technology provider with the strong presence and strategic location of academia, chemical industries and other stakeholders in the value chain. In the Port of Rotterdam in the Netherlands about 5 million tons of aromatic building blocks are currently produced and distributed to the chemical industry in the Netherlands, Germany, Belgium and other countries. These aromatic bulk chem­icals used and produced consist of so-called aromatic monomers like BTX, styrene and phenol.

In the Netherlands in 2010 the Wageningen UR Lignin Platform was established, which plays an impor­tant role in this lignin valorization value chain develop­ment (http://www. wageningenur. nl/Lignin-Platform. htm). This is a joint research program with academia and industry dedicated to develop the entire lignin bio­aromatics value chain. Besides this initiative other net­works in Canada and Scandinavia work on lignin valorization topics.

Considering these increased research activities on lignin conversion and valorization technologies it can be concluded that the race to produce bioaromatics from renewable feedstocks is wide open. Next to lignin as aromatic feedstock conversion of carbohydrates to aromatic chemicals is also under investigation (Dodds and Humpheys, 2013). It should also be emphasized that many of the above-discussed technologies are at a very early stage, which makes it at present unclear if and which of those routes can become cost competitive as well as sustainable.

CONCLUSIONS AND FURTHER
PERSPECTIVES

The use of lignocellulosic feedstocks as an important source for chemicals and fuels is gaining momentum. This chapter has indicated that there are many vari­ables to take into consideration. We have learned that lignocellulosic biomass consists of three major groups: the softwoods, hardwoods and grasses and that there is also great heterogeneity within each group. There are multiple pretreatment routes developed that are currently scaled up to pilot, demonstration and com­mercial scales. The optimal pretreatment technology needs to be selected based on the available feedstock and the desired product. At the moment there are no indications that one pretreatment method will be the optimal route for all feedstocks and products. Many routes toward chemical building blocks based on monomeric carbohydrates are ready for scaling up; lignin conversion into monomeric building blocks needs substantial additional R&D before economical processes are within reach.

Productions of biofuels (bioenergy processes) use biobased (sustainable) feedstocks, and convert energy — latent plant or algal molecules (formed by photosyn­thesis) to molecules more suited or amenable as fuels (see chapters 10,11,15,18—20 of this book). The key in­termediate, platform molecules of bioenergy processes, such as glucose for starchy or cellulosic bioethanol production, may be used to produce nonfuel biochemi­cals, such as organic acids, polyols, polymers or plastics (Clark et al., 2012; Dapsens et al., 2012; Koutinas et al., 2007, 2006). As the economics and scale of biofuel and biochemical productions continue to grow, it becomes more important to enhance the values of various co — and by-products, so that the biofuel or biochemical pro­duction can be upgraded or expanded to a more efficient biorefinery capable of maximally utilizing biobased feedstocks. Such expansion is desirable considering the colocation and energy/material source sharing, as well as the integrability of the existing phytochemical production technologies (mentioned in Section (Produc­tion of Industrial Phytochemicals)), with the bioenergy processes. Many approaches might be taken to recover phytochemicals during bioenergy or biochemical production processes. For instance, an upstream frac­tionation (e. g. dry milling or air classification) might be added to allow further processing of crudely sepa­rated feedstock components.

Coproduction from Starch — or Sugar-Based Bioenergy Processes

Starch — or sugar(cane)-based bioethanol processes are fully commercialized, and are the major bioethanol pro­viders at present. The main feedstocks are corn and sugarcane, and to a less extent, potato, cassava and sugar beet. Starch is converted by amylolytic enzymes to fermentable sugars (mainly glucose), sucrose is squeezed out from cane or beet, and sugars are fer­mented by yeast to ethanol. For corn ethanol processes, the main coproducts are DDGS for feed, as well as steep liquor, gluten meal, corn oil, and fiber from wet milling (Zhang et al., 2012, Figure 20.6). Main by-products are corn stover and cob for corn ethanol processes, and bagasse for sugarcane ethanol process. These by­products are currently being developed as feedstocks for lignocellulosic ethanol. Many phytochemicals of industrial interest might be obtained or derived from the co — or by-products of starch — or sugar-based bio­energy processes, as exemplified in Section (Coproduc­tion from Processing (Biorefinery) of Staple Crops). Obtaining betaine from sugar beet has been shown (Kripp, 2006). It has also been reported that polyolefins (e. g. polypropylene and polyethylene), polymerized polyurethane or other biomaterials may be made from DDGS (Diebel et al., 2012, Tatara et al., 2007; Chees — brough et al., 2008).

For many fuel purposes, alkanes are more desirable than the other biofuels already discussed. For example, jet fuel standards (Jet-A or JP-8) demand a fuel with high energy density, low viscosity, low freezing point and good physical-chemical compatibility. These criteria cannot be met with fuels such as ethanol or fatty acid methyl esters, biodiesel. Being able to directly make al­kanes would have a great payoff as these biofuels are "drop-in" fuels, able to directly substitute for presently used petroleum-based fuels as they could be used with existing infrastructure and would require no engine modification, etc.

Cyanobacteria, like some other bacteria, have long been recognized as being able to synthesize at least very small quantities of alkanes, which in fact can serve as a biogeochemical marker for their presence in the past (Han et al., 1968; Winters et al., 1969). This was taken advantage of in a recent demonstration of the heterotro­phic production of alkanes using a modified E. coli that expressed the alkane biosynthetic pathway from a cyanobacterium, consisting of an acyl-carrier protein reductase, which produces a fatty aldehyde, and an aldehyde decarbonylase (Schirmer et al., 2010). This allowed the production and secretion of a variety of C13—C17 alkanes and alkenes. Of course it would be desirable to actually do this in a cyanobacterium, and one study examined this through the heterologous expression of fatty acyl-CoA reductase in Synechocystis (Tan et al., 2011), which allowed the production of small quantities of aliphatic alcohols. The acc genes, encoding acetyl-CoA carboxylase (ACCase), which catalyses what is believed to be the rate-limiting step of fatty acid biosynthesis, were introduced into the genome in hopes of boosting alkane production, but only insignificant quantities were made. Further work is required to demonstrate significant alkane synthesis by a cyanobac­terium. However, it may prove difficult to greatly boost alkane synthesis in this oxygen-evolving organism as the critical enzyme, aldehyde decarbonylase, has recently been shown to be a di-iron enzyme with an unusual mechanism that requires anaerobic conditions for full activity (Das et al., 2011).

CONCLUSION AND OUTLOOK

As discussed in this chapter, recent studies have shown the great promise for biofuels production by cyanobacteria. Unique among possible biofuel producers, cyanobacteria combine the attributes of being able to carry out photosynthesis-driven carbon dioxide fixation and to be easily manipulated geneti­cally. The next few years should see advances in increasing the production rates and titers of the different demonstrated biofuels as well as perhaps the widening of the spectrum of possible biofuels. Nevertheless, for cyanobacterial systems to live up to their potential, a number of serious hurdles must be overcome. These include the development of reliable methods of stable cyanobacterial mass culture at high levels of productivity and the demonstration of cost — effective harvesting strategies. Harvesting presents a real dilemma no matter what the biofuel. If the biofuel is contained within the cell, then the biomass has to be removed from the culture medium, of which it is less than 1% by weight. If the biofuel is an excreted liquid, then this will necessarily be quite dilute and require substantial concentration. If the biofuel is a gaseous product, the culture will have to be enclosed in air­tight transparent material at a substantial cost given the large surface areas that would be required. Of course, the payoff to solving these problems would be enormous and this is likely to inspire future research and development in this area.

The pretreatments described above such as steam explosion, liquid hot water, dilute acid, lime, and ammonia pretreatments are the most studied methods because they have potential as cost-effective pretreat­ments (Kazi et al., 2010; Mosier et al., 2005; Piccolo and Bezzo, 2009; Tao et al., 2011; Wyman et al., 2005). Other alternatives such as biological, ultrasonication, micro­wave, organosolvs, ILs, and combinatorial methods are also essayed; however, they are either low effective, long-time treatment or too expensive, and further inves­tigation and improvements have to be reached before they can be competitive. In this section multiple or combinatorial pretreatments and other alternative pretreatments will be discussed.

Biological pretreatments must decrease the time of the process in order to be competitive in an industrial concept of biorefinery; to reach this objective its combi­nation with chemicals and/or physical methods has been proposed by several studies. For example, the combination of a biological pretreatment by I. lacteus or P. subvermispora with a mild alkali pretreatment improved significantly ethanol production without the production of inhibitor compounds for downstream pro­cesses (Salvachua et al., 2011; Zhong et al., 2011). Other two-step pretreatment proposed consisted in a mild physical or chemical step (ultrasonic and H2O2) and a subsequent biological treatment by P. ostreatus, increasing significantly the lignin degradation compared to those of one-step pretreatments (Yu et al., 2009); also, pretreat­ment by white-rot fungi has been combined with organo — solv pretreatment in an ethanol production process from beech wood chips (Salvachua et al., 2011); the combina­tion of biological and mild acid pretreatment was reported as a promising method to improve enzymatic hydrolysis and ethanol production from water hyacinth with low lignin content (Ma et al., 2010). Another combi­nation of biological pretreatment with thermal processing for wheat straw consisted in a first phase of biodegrada­tion by P. chrysosporium (10 days) and a thermal decom­position using pyrolysis (Zeng et al., 2011). Also, a combination of fungal treatment with liquid hot water treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa (Wang et al., 2012).

Sugarcane bagasse is one of the most promising biomass considered in biorefineries; thus, several studies have proposed combined pretreatments. The ultrahigh-pressure explosion combined with alkaline treatment (0.5% NaOH) at 125 °C for 120 min signifi­cantly decreased the particle size and disrupted the microstructure, with a significant delignification and increased enzymatic digestibility of sugarcane bagasse (Chen et al., 2010). A combined treatment with dilute sulfuric acid and microwave heating up to 190 °C for 5 min has also been studied. This treatment resulted in an increment of the specific surface area of bagasse, almost complete removal of hemicellulose and signifi­cant reduction of the crystalline structure of cellulose (Chen et al., 2011), while microwave—alkali treatment at 450 W for 5 min resulted in almost 90% of lignin removal from the bagasse (Binod et al., 2012). Also, bagasse has been subjected to sono-assisted alkaline pre­treatment (Velmurugan and Muthukumar, 2012a). Acid, alkaline or sequential acid/alkaline solutions have been tested to conversion into bio-oil in a pyrolysis process at low-temperature conversion under He or O2/He atmo­spheres at 350—450 °C (Cunha et al., 2011). A two — stage process for delignification of sugarcane bagasse uses alkali and peracetic acid combination (Teixeira et al., 2000; Zhao et al., 2011b).

Same strategies (acidic/alkaline) have been proposed for corn stover. For example, a two-stage process consists of use of 0.07 wt% sulfuric acid at 170 °C,

2.5 ml/min for 30 min and ARP (15 wt% ammonia) at the same temperature, 5.0 ml/min for 60 min. In the first stage hemicellulose was recovered while in the following stage lignin was recovered. This treatment brought about enzymatic digestibility of 90% using 60 filter paper units/g glucan cellulase enzyme loadings (Kim, 2011). Another combined treatment proposed for corn stover is SAA (15 wt% ammonia) with solution con­taining also 20 wt% ethanol at 60 °C for 24 h preserving the hemicellulose in solid form (Kim et al., 2009). Also, the use of NaOH (0.3 N) and a step of particle size homogenization has reported a significant enhancement of enzymatic hydrolysis (Li et al., 2004). The synergistic effect of preimpregnation by sulfuric acid (3 wt%) and steam explosion (190 °C) has been investigated; after 48 h of digestion the yield of glucose was 93% of the theoretical (Zimbardi et al., 2007).

Sequential stages of autohydrolysis and ethanol— water mixtures were used to pretreat olive tree trimmings recovering up to 42% of the polysaccharides contained in the raw material (Requejo et al., 2011). Also, this process has been tested with uncatalyzed ethanol—water solu­tions of Eucalyptus globulus wood (Romani et al., 2011). Mixtures of ethanol/water/acetic acid in an autoclave have been also used (Teramoto et al., 2008). This combined process causes the solubilization of hemicelluloses and lignin, leaving solids enriched in cellulose. A treatment of ethanosolv catalyzed with FeCl3 (0.1 M) at 170 °C for 72 h has been proposed for barley straw allowing enzy­matic digestibility of 89%. This treatment had a particu­larly strong effect on enzymatic digestibility and cellulose recovery (Kim et al., 2010).

Another pretreatment at pH 1 (hydrochloric acid) and subsequently at pH 13 (sodium hydroxide) released 69% and 95% of the theoretical maximal amounts of glucose and xylose, respectively, from the straw and removal of 68% of the lignin (Pedersen et al., 2010). The opposite sequence alkaline stage (ammonia) followed by acidic stage (dilute sulfuric acid by percolation) has also been used to treated rice straw (Kim et al., 2011a).

Microwave-based heating (190 °C) was used to pre­treat switchgrass presoaked in alkali solutions (0.1 g/g) resulting in release of 90% of maximum potential sugars. This value was significantly higher than the one obtained with conventional heat and it was attributed to the disruption of recalcitrant structures under microwave heating (Hu and Wen, 2008).

Significant disintegration of lignocellulosic struc­ture of wheat, barley straw grinds, switchgrass and coastal bermuda grass has been reported with the microwave—chemical (NaOH or Ca(OH)2) pretreat­ments (Kashaninejad and Tabil, 2011; Keshwani and Cheng, 2010). Also, microwave-assisted pretreatment of woody biomass with ammonium molybdate acti­vated by H2O2 has also been proposed resulting in a selective delignifying system (Verma et al., 2011).

For hydrogen production from Miscanthus by Ther­motoga elfii, high delignification values were obtained by the combination of mechanical (one-step extrusion) and chemical pretreatments (NaOH at 70 °C) resulting in a 33% conversion into monosaccharides of the initial biomass after enzymatic hydrolysis (de Vrije et al.,

2002) .

A two-stage pretreatment method was proposed and tested for deconstruction of Miscanthus; first, biomass is pretreated at 50 °C, 1.0—4.0% alkaline peroxide solutions to remove up to 64% of hemicellulose and 64% of lignin. The remaining solids were subjected to a second pre­treatment at 121 °C with electrolyzed water (Wang et al., 2010).

On the other hand, application of a dehydration pro­cess to the mechanochemical pretreatment process of the bioethanol production system has been proposed for energy saving and cost reduction. However, the dehy­dration process has problems with the loss of sugars eluted in the liquid phase during the hydrothermal pro­cess (Yanagida et al., 2011).

Combination of hot compressed water (hydrothermal treatment) and mechanochemical milling, including a dewatering step for Eucalyptus and rice straw, has been proposed for ethanol production (Fujimoto et al., 2008; Hideno et al., 2012). Torrefaction is a mild thermal pre­treatment (T < 300° C) that improves biomass milling and storage properties (Chen et al., 2012; Fisher et al., 2012). This treatment has gained attention in recent years and some biomasses that have been treated include oil palm fiber and eucalyptus, Norwegian birch, spruce, Miscanthus and white oak sawdust; residues from coffee grain, sugarcane, sawdust and rice husk bagasse (Chen et al., 2012; Lu et al., 2012; Medic et al., 2012; Protasio et al., 2012; Srinivasan et al., 2012; Tapasvi et al., 2012; Tumuluru et al., 2012). Wet torrefaction (hot compressed water 200—260 °C) and dry (nitrogen, 250—300 °C) has been tested with Loblolly pine with mass yield of solid product ranging between 57% and 89%, and energy densification to 108—136% of the orig­inal feedstock (Yan et al., 2009).

Extrusion has also been used in combination with alkali (1.70%, w/v NaOH) soaking for pretreatment of prairie cord grass at a barrel temperature of 114 °C, 122 rpm screw speed resulted in an 82% of sugar recov­ery after enzymatic hydrolysis (Karunanithy and Muthu — kumarappan, 2011). An alkali-combined extrusion pretreatment of corn stover obtained glucose and xylose sugar yields of 86.8% and 50.5%, respectively. The condi­tions used were alkali loading of 0.04 g/g dry biomass, a screw speed of 80 rpm, residence time for extrusion is 27 min, temperature of 140 °C and washed with water (Zhang et al., 2012b). Also, glucose conversion of 95% was reported from soybean hulls using a thermomechan­ical extrusion pretreatment (screw speed 350 rpm, 80 °C and in-barrel moisture content 40% wt) (Yoo et al., 2011). A study of high-temperature (110—130 °C), concentrated — acid (5—30 wt.%) hydrolysis kinetics was undertaken for pretreated pine in a corotating twin-screw extruder reactor, obtaining more than 50% of the theoretical glucose in roughly 25 min (Miller and Hester, 2007). A successive pretreatment of ball-milled bamboo con­sisted in ultrasound treatment in ethanol solution at 20 ° C from 0 up to 50 min. After that the samples were dissolved with 7% NaOH/12% urea solutions at 12 °C, followed by successive extractions with dioxane, ethanol, and dimethyl sulfoxide (Li et al., 2010). Other treatments, such as SAA and proton beam irradiation, have been tested with rice straw and approximately 90% of the theoretical glucose conversion was obtained at 12 h (Kim et al., 2011b). Microwave pretreatment also has been combined with alkali to pretreat cashew apple bagasse founding that alkali exerted influence on glucose formation (Rodrigues et al., 2011).

A pretreatment method using ammonia and ILs reported a synergy effect for rice straw, achieving 82% of the cellulose recovery and 97% of the enzymatic glucose conversion with recycling of the ILs (Nguyen et al., 2010). Pretreatment of wheat straw with combined sulfuric acid (0—3%, w/v) and Tween-20 (concentration,

0— 1%) was evaluated with modification of lignin surface (Qi et al., 2010). Other surfactants, such as, Tween-80, dodecylbenzene sulfonic acid, and polyethylene glycol 4000, have also been used combined with diluted acid to treat corn stover and bagasse (Qing et al., 2010; Sindhu et al., 2012).

Other pretreatments include technology used in kraft pulp mills for the efficient conversion of lignocellulosic biomass into ethanol (Gonzalez et al., 2011). Sulfite pre­treatment to overcome recalcitrance of lignocellulose consists of sulfite treatment of wood chips under acidic conditions followed by mechanical size reduction using disk refining (Li et al., 2012; Zhang et al., 2012a). Pretreat­ment of corn stalk with sulfite (7%) at a temperature of 180 °C for 30 min was successfully performed (Liu et al., 2011; Zhu et al., 2009). Silage preparation is a well-known procedure for preserving plant material; the effects of Fe(NO3)3 pretreatment conditions on sugar yields were investigated for corn stover silage. Ensiling techniques, with and without supplemental enzymes, also have been reported as a cost-effective pretreatment (Chen et al., 2007; Sun et al., 2011; Thomsen et al.,

2008) . Also, FeSO4 (0.1 mol/L at 180 °C for 20 min) was investigated as a catalyst for the pretreatment of corn sto­ver, observing significantly increased hemicellulose degradation in aqueous solutions with high xylose recovery and low cellulose removal (Zhao et al., 2011a). Lignocellulose pretreatment featuring modest reaction conditions (50 °C and atmospheric pressure) was demon­strated to fractionate lignocellulose to amorphous

cellulose, hemicellulose, lignin, and acetic acid by using a nonvolatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone) and water (Zhang et al., 2007).

• Requires high reaction temperatures.

• Soap formation: in the base-catalyzed transesterification process, free fatty acid (FFAs) level of feedstock should be less, otherwise it will result in too much soaps formation.

• Recovery of by-product: purification of glycerol is very difficult.

• Pretreatment step needed: FFAs level of the feedstocks should not exceed 3 wt%, beyond which it has to undergo pretreatment steps before transesterification (Leung et al., 2010).

• Yield of methyl esters: yields of the methyl esters are lower compared to enzymatic transesterification.

• Purification of methyl esters: purification of methyl esters requires repeated washing which increases process operational cost.

• Less active: since alkali catalysts (NaOH and KOH) are inexpensive, they are preferred but activity is less (Demirbas, 2008).

• Energy consumption: alkali-catalyzed transesterification needed large energy consumption during downstream biodiesel refining process (Madras and Kolluru, 2004).

• Corrosion: when H2SO4 is used as catalyst, it leads to corrosion of the reactor and huge wastewater generated during neutralization of mineral acid (Atadashi et al., 2013).

• Use of homogenous catalysts makes biodiesel product separation difficulty and recovery of catalyst cumbersome (Atadashi et al., 2013).

• Acid-catalyzed transesterification reaction needs higher alcohol-to-oil molar ratios (Atadashi et al., 2013).

• In base-catalyzed transesterification reaction, large amount of catalyst is needed.

Difficulties arise during chemical catalysis can be overcome by enzyme-mediated (biocatalysts) transester­ification and they are becoming increasingly important in biodiesel preparation due to their ability to beat chem­ical catalysts. Lipases (E. C.3.1.1.3) are widely considered as biocatalysts to catalyze transesterification and esteri­fication reactions.

India ranks seventh as the world’s energy producer accounting for about 2.5% of the world’s total annual energy production, and world’s fifth largest energy consumer with about 3.5% of the global primary energy demand (IEA, 2007; Planning Commission, Govt. of India, 2007). Despite being among the largest energy producer, India is a net importer of energy, largely due to huge imbalance between energy consumption and production. About 30% of India’s total primary energy need is being met by petroleum oil, of which 76% is imported. India’s transportation fuel require­ments are unique in the world. India consumes almost five times more diesel fuel than gasoline, whereas all other countries in the world use more gasoline than diesel fuel (Khan et al., 2009). Thus, search for alterna­tives to diesel fuel is of special importance in India. Bioalcohols are unsuitable substitutes for diesel engines, because of their low cetane numbers (CNs) along with poor energy content per unit biomass (Bhattacharyya and Reddy, 1994; Rao and Gopalkrishnan, 1991). There­fore, biodiesel is the only option to fulfill the require­ments in future.

BIODIESEL

Biodiesel is chemically monoalkyl esters of long — chain fatty acids derived from vegetable oils or animal fats. The history of using vegetable oil as an alternative fuel dates back to 1900, when Rudolph Diesel used pea­nut oil as fuel in the World Exhibition in Paris. It was found that vegetable oils, in general, have acceptable CNs and calorific values comparable with the conven­tional diesel. However, the major problem with the direct use of vegetable oils as fuel of compression igni­tion engine is their high viscosity, which interferes with the fuel injection and atomization contributing to incomplete combustion, nozzle clogging, injector coking, severe engine deposits, ring sticking and gum formation leading to engine failure (Knothe, 2005; Meher et al., 2006; Singh and Rastogi, 2009). Therefore, vegetable oils need to be modified to bring their combustion-related properties closer to those of diesel fuel. One possible method to overcome the problem of high viscosity of vegetable oils is their chemical modification to esters, what is nowadays called as "biodiesel".

Biodiesel has emerged as the most suitable alterna­tive to petroleum diesel fuel owing to its ecofriendly characteristics and renewability (Krawczyk, 1996). It burns in conventional diesel engines with or without any modifications while reducing pollution (100% less sulfur dioxide, 37% less unburned hydrocarbons, 46% less carbon monoxide, and 84% less particulate matter) in comparison to the conventional diesel fuel (McMillen et al., 2005). The basic feedstocks for the production of first-generation biodiesel were mainly edible vegetable oils like soybean, rapeseed, sunflower and safflower. The use of first-generation biodiesel has generated a lot of controversy, mainly due to their impact on global food markets and food security for diverting food away from the human food chain. The second-generation biodiesel was produced by using nonedible oil sources like used frying oil, grease, tallow, lard, karanja, jatropha and mahua oils (Alcantara et al., 2000; Francis and Becker 2002; Canakci and Gerpen, 1999; Dorado et al., 2002; Ghadge and Raheman, 2006; Mittelbach, 1990). Nevertheless, the cost of biodiesel production is still a major obstacle for large-scale commercial exploitation, mainly due to the high feed cost of vegetable oils (Lang et al., 2001). Moreover, the first- as well as the second-generation biodiesel based on terrestrial plants initiate land clearing and potentially compete with net food produc­tion (Chisti, 2008; Marsh, 2009). The focus of researchers has now been shifted to the next generation biodiesel. The third-generation biodiesel is both promising and different; it is based on simple microscopic organisms that live in water and grow hydroponically, i. e. microalgae.

The possibilities of biodiesel production from edible oil resources in India is almost impossible, as primary need is to first meet the demand of edible oil that is being imported. India accounts for 9.3% of the world’s total oil seed production and contributes as the fourth largest edible oil producing country. Even then, about 46% of edible oil is imported for catering the domestic needs (Jain and Sharma, 2010). So the nonedible oil resources like Jatropha, pongamia, mahua, etc. seem to be the only possibility for biodiesel production in the country. The Government of India has duly realized the impor­tance of biodiesel and introduced a nationwide program under the National Biodiesel Mission in 2003 with the aim of achieving a target of meeting 13.4 Mt of biodiesel (@ 20% blending) from Jatropha curcas by 2012, and to achieve the target about 27 billion of planting materials are required to be planted over 11.2 million hectares of land (Planning Commission, Govt. of India, 2003). At the current rate of consumption, if all petroleum — derived transport fuel is to be replaced with biodiesel from Jatropha oil, Jatropha would need to be grown over an area of 384 million hectares, which is more than 100% of the geographic area of India (Khan et al.,

2009) . Therefore, India must find additional, reliable, cost-effective and sustainable feedstock for biodiesel production. In this context, biodiesel from microalgae seems to be a suitable substitute for diesel fuel in the long run.