The process of isolation and selection of algae strains needs to consider the requirements of algal oil suitable for biodiesel production. Algal lipids oc­cur in cells predominantly as either polar lipids (mostly in membranes) or lipid bodies, typically in the form of triacylglycerides. The latter are accu­mulated in large amounts during photosynthesis as a mechanism to endure adverse environmental conditions. Polar lipids usually contain polyunsatu­rated fatty acids which are long-chained, but have good fluidity properties. TAG in lipid storage bodies typically contain mostly saturated fatty acids which have a high energy contents, but, depending on the fatty acid profile of the algae strain, may lack fluidity under cold conditions. Provided the algal oil is low enough in moisture and free fatty acids, biodiesel is typical­ly produced from TAG with methanol using base-catalyzed transesterifica­tion [12]. Most current feedstock for biodiesel production is based on plant oils produced from oil palm, soybean, cottonseed and canola, recycled cooking greases or animal fats from beef tallow or pork lard [13]. Ac­cording to Fukuda, transesterification using base catalysts is 4,000 times faster than using acid catalysts [14]. Some common base catalysts used by industry are sodium hydroxide and potassium hydroxide. Use of lipase enzymes as a catalyst is efficient, however their use is limited because of the high costs [14]. The best temperature for the reaction is typically 60 °C under normal atmospheric pressure. If the temperature is higher, metha­nol will boil, lowering the efficiency [14]. During the transesterification process, saponification reactions can occur, forming soap. Thus, oil and alcohol must be dried. Finally, biodiesel is recovered by washing rapidly with water to remove glycerol and methanol [4]. The high potential of oil production from microalgae has attracted several companies to com­mercialize biodiesel from microalgae (e. g., MBD Energy Pty and Muradel Pty Ltd in Australia). Basically, algal biodiesel is produced after algae cultivation and harvesting, followed by oil extraction and its conversion by transesterification. Principally, microalgal oil can be directly used as fuel feedstock, based on the conventional process of biodiesel production, provided the fatty acid profile is favorable. But even algal oils with a high degree of saturation (e. g., similar as tallow) can be considered as a drop — in fuel (e. g., for B20 blends). In addition, scientists are also focusing on the conversion to higher value products. For example, thermal cracking is used for decomposition of triglycerides into hydrocarbons such as alkans, alkenes, and aromatic compounds [15,16].

Thermochemical conversion covers different processes such as direct combustion, gasification, thermochemical liquefaction, and pyrolysis. In pyrolysis, heating rate affects reaction rate which in turn affects product composition. Traditional heating methods require expensive heating mech­anisms to achieve rapid temperature rise with poor process control. Micro­wave assisted pyrolysis (MAP) has following advantages: fine grinding of biomass is not necessary; microwave heating is mature and scalable technology which is suitable for distributed biomass conversion. Due to insufficient understanding of the mechanism of pyrolysis and the lack of effective control of the pyrolysis process, pyrolytic bio-oils are complex mixture with low calorific value, high acidity, high oxygen volume, and poor stability. However, bio-oil from pyrolysis of microalgae appears to have higher quality than those from cellulosics [52].

In recent years, the role of catalyst and minerals in biomass pyrolysis was investigated. Lu et al. [53] reported that ZnCl2 could catalyze fast pyrol­ysis of corn cob with the main products of furfural and acetic acid. Du et al. [54] used 1-butyl-3-methylimidazolium chloride and 1-butyl-3-methylimid — azolium boron tetrafluoride as the catalysts in MAP of straw and sawdust.

It is well known that many strains of algae are capable of growing in wastewater and by doing so providing a form of treatment. Outdoor pro­ductivities are, however, difficult to find in the literature as most studies are performed in the laboratory and therefore conditions are less realis­tic. Likely strains to dominate in specific scenarios and locations are not known and therefore it is hard to speculate what type of strain would be dominant. It is therefore also difficult to know what harvesting technique would be most appropriate for the strain cultivated and how much energy could be expected to be recovered from conversion to biodiesel, bioetha­nol, biogas or from combustion.

Each of the processes studied have been tested and are considered practically viable. Each of the harvesting methods considered are cur­rently used for harvesting algal biomass regardless of their energy use and overall viability. Conversion techniques have been shown to be feasible, again regardless of how viable they are in real situations. What are missing are pilot-scale studies of the whole system to give valuable information about applicability to different situations.

The WCI is calculated using Equation (15). Figure 3 plots the second — order water intensity of transportation (consumption and withdrawal) us­ing algal biofuels produced in this system (bio-oil and methane) for the two cases considered. These data are shown alongside equivalent results for a variety of transportation fuels, including fossil fuels, electricity for electric vehicles, and biofuels reported previously by King and Webber [33] (note the logarithmic scales).

As shown, the Experimental Case water intensity (which includes sig­nificant research-scale artifacts, no recycling, evaporation from the ponds, and relatively low biofuel yields) far exceeds any of the other transpor­tation fuels. Meanwhile, the Highly Productive Case water consumption intensity is lower than that of biofuels from irrigated crops, while its water withdrawal intensity is similar to, or slightly greater than, that of biofuels from irrigated crops. Still, the Highly Productive Case, which assumes very efficient water use (no evaporation and 95% recycling), is much more water intensive than traditional fossil fuels or non-irrigated biofuels from conventional feedstocks. While the WCI and WWI metrics are useful to evaluate the magnitude of water required for fuel production, they do not consider water quality (that is, algae can be grown in degraded, brackish, or saline sources, for which the concerns about water quantity are muted as compared with freshwater). The relationship between water require­ments (considering magnitude and quality) and water availability (includ­ing precipitation, which is not considered here for the algal biofuel cases) is more important than the water intensity, alone. However, this relation­ship is dependent on location and must be evaluated on a case-by-case, site-specific basis for all of the fuels shown.

Several other studies have been conducted to determine the water in­tensity of algal biofuel production and the system boundaries used in each study vary [9,11,15,17,54,55]. Analogous to energy inputs, the water in­puts for a production pathway include direct and indirect parts. Addition­ally, the water consumption required to produce capital equipment can be included (e. g., water required for producing glass bioreactors [54]). Finally, the water intensity is dependent on co-product allocation, as the

FIGURE 3: Second-order water intensity of transportation for several fuels [33], including the bio-oil and methane co-products from the two algal biofuel cases: the Experimental Case and the Highly’ Productive Case. *Note the logarithmic scale. To evaluate sustainability, the water intensity and required water quality must be considered in conjunction with water availability.

total water consumed to operate the production pathway should be allo­cated between the bio-oil and co-products (e. g., methane).

The WWI is calculated according to Equation (16). The WWI for the Experimental Case and the Highly Productive Case are 87,000 L/km and 220 L/km, respectively. Like the nutrient analysis presented below, the water analysis underscores the advantages of using nutrient-rich low-qual­ity water, like waste water or agricultural runoff [28]. In these cases, the incremental water usage is minimized and the discharge water can be of higher quality (e. g., higher purity) than the water input.

JUAN CARLOS SERRANO-RUIZ, ANTONIO PINEDA,

ALINA MARIANA BALU, RAFAEL LUQUE,

JUAN MANUEL CAMPELO, ANTONIO ANGEL ROMERO, and JOSE MANUEL RAMOS-FERNANDEZ

7.1 INTRODUCTION

Fossil fuels are the primary source of energy, chemicals and materials for our modern society. Petroleum, natural gas and coal supply most of the energy consumed worldwide and their massive utilization has al­lowed our society to reach high levels of development in the past cen­tury. However, these natural resources are highly contaminant, unevenly distributed around the world and they are in diminishing supply. These important concerns have stimulated the search for new well-distributed and non-contaminant renewable sources of energy such as solar, wind, hydroelectric power, geothermal activity, and biomass. This shift toward a renewable-based economy is currently spurred by governments which have established ambitious targets to replace an important fraction of fos­sil fuels with renewable sources within next 20 years [1] and [2]. In this sense, biomass is considered the only sustainable source of organic carbon currently available on earth and, consequently, it is the ideal substitute for

petroleum in the production of fuels, chemicals and carbon-based materi­als [3].

Transportation sector of our society heavily relies on petroleum which accounts for essentially all (96%) of the transportation energy. This high reliance on petroleum is especially relevant since transportation is the largest and fastest growing energy sector, and it is responsible for almost one third of the total energy consumed in the world [4]. A large fraction of the extracted petroleum (70-80%) is consumed in form of transporta­tion fuels (e. g. diesel, gasoline and jet fuels) in an attempt to cover this enormous demand for transportation energy. Consumption of petroleum is currently estimated to be around 80 millions of barrels per day, with pro­jections to increase this amount by 30% within the next 20 years [4]. With these aspects in mind, an eventual displacement of petroleum by biomass will necessarily involve development of new technologies for large-scale production of fuels from this resource, the so-called biofuels.

The liquid biofuels most widely used today are bioethanol and bio­diesel which are obtained from edible biomass sources such as sugar cane or corn and vegetable oils, respectively. An exponential increase in the consumption of such biofuels has taken place in the past few years [5]. Two are the main driven forces for this rapid expansion of bioethanol and biodiesel: (i) the simple and well-known technologies for their production (e. g. fermentation of sugars and transesterification of triglycerides with methanol) that has accelerated scale up of technologies and subsequent commercialization; and (ii) the partial compatibility of these biofuels with existing transportation infrastructure of diesel and gasoline which has al­lowed an easy penetration of these biofuels in the current fuel market.

Even though biodiesel and bioethanol (denoted as conventional bio­fuels) are produced by simple and mature technologies and are already commercially available, they possess a number of important drawbacks that seriously limit their further implementation in current transportation infrastructure [6]. For example, bioethanol is slightly corrosive and it has to be used in form of dilute mixtures with gasoline (e. g. E blends) in exist­ing spark-ignition vehicles; it contains less energy per volume than gaso­line (leading to lower fuel economy of vehicles running on E blends); and it induces water absorption in the fuel when added to gasoline thereby increasing risk of phase-separation episodes and engine damages. The corrosive nature of biodiesel also obligates to use it dilute with petrol-based diesel (e. g. B blends) and its higher cloud point compared to regular diesel in­creases the risk of plugging filters or small orifices at cold temperatures. Furthermore, this issue is compounded for the new generation of diesel engines which operate at higher injection pressures and with nozzles with a lower diameter.

These important limitations of conventional biofuels have stimulated the search for new technologies that allow production of high energy-den­sity, infrastructure-compatible fuels (i. e. advanced biofuels) which could be easily implemented in the existing hydrocarbons-based transportation infrastructure (e. g. engines, fueling stations, distribution networks and pet­rochemical processes). In the past few years, these strong incentives have favored a dramatic change in funding directions from projects involving biodiesel and bioethanol to those aimed to the synthesis of advanced bio­fuels [7]. Relevant examples of advanced biofuels include higher alco­hols (C4-C7) which possess energy density and polarity properties similar to gasoline [8]; and liquid hydrocarbon fuels (e. g. green hydrocarbons) which are chemically identical to those currently used in the transportation fleet [6], [9] and [10].

When operating with biomass resources, the structural and chemical complexity of feedstocks is an important issue. One of the most common strategies to overcome biomass complexity involves previous conver­sion into simpler fractions that are more easily transformed in subsequent processes. Thus, complex biomass resources can be converted into sim­pler compounds or platform molecules, which can subsequently serve as starting materials for a number of valuable products [11]. These platform molecules are carefully selected in base of a number of indicators such as the availability of commercial technologies for their production from biomass sources, and the platform potential of these compounds for the simultaneous production of fuels and chemicals in biorefineries [11]. This selected group of biomass platform molecules include sugars (glucose, xylose), polyols (sorbitol, xylitol, glycerol), furans (hydoxymethyl furfu­ral or HMF, furfural), acids (lactic acid, levulinic acid, succinic acid) and alcohols (ethanol).

In the present paper we explore the potential of two of these platform molecules (e. g. lactic acid and levulinic acid) for the production of advanced biofuels. As represented in Fig. 1, the very different chemical composition of these molecules compared to final products (e. g. hydrocarbons or higher alcohols) suggests that deep chemical transformations will be required dur­ing catalytic processing of these resources. As is common to all biomass derivatives, lactic acid (2-hydroxypropanoic acid) and levulinic acid (4-oxo — pentanoic acid) are highly oxygenated compounds and, consequently, their conversion into advanced biofuels will necessarily involve deoxygenation steps. This oxygen removal step increases energy density in the molecule and, simultaneously, achieves reduction of the chemical reactivity generating less-reactive intermediates that are more easily processed to final products with high yields. As a result, oxygen will be removed from biomass acids in form of H2O and/or COx species (CO and CO2) by hydrogenation, dehydra­tion, Csingle bondO hydrogenolysis and decarboxylation/decarbonylation catalytic processes. This requisite deoxygenation step normally (although not always) involves consumption of large amounts of hydrogen which is expensive and typically derived from fossil sources (which negatively af­fects CO2 footprint of the bioprocess). As will be shown in subsequent sec­tions, efforts are currently being made to drastically reduce external hydro­gen consumption during deoxygenation of biomass platform molecules by, for example, utilization of renewable sources of this gas such as formic acid (a by-product of levulinic acid production industrial process) [12] and [13].

The high oxygen content of biomass platform molecules (as compared to petroleum feedstocks) is not the only limitation to overcome when ad­vanced fuel production technologies are envisaged. Platform molecules are typically derived (by chemical and biological routes) from biomass sugars which are compounds with a maximum number of carbon atoms limited to 6 (derived from glucose). Consequently, if targeted products are liquid hydrocarbon transportation fuels (e. g. C5-C12 for gasoline, C9-C16 for jet fuel, and C10-C20 for diesel applications) deoxygenation will necessarily have to be combined with additional reactions aimed to increase the molecular weight in the molecule (e. g. Csingle bondC cou­pling reactions) [14]. Among the numerous Csingle bondC bond forming routes that organic chemistry can offer us, there are some reactions with particular interest in biomass conversion processes [15] and [16]. Thus, well known reactions such as aldol condensation of carbonyl compounds, catalytic ketonic decarboxylation or ketonization of carboxylic acids,

C0X, H20

H2

FIGURE 1: Scheme of the catalytic routes required to convert biomass-derived acids such as lactic acid and levulinic acid into advanced biofuels.

oligomerization of alkenes, and alkylations of hydrocarbons are especially indicated to increase molecular weight and to adjust structure of final ad­vanced biofuel products.

Aldol condensation and ketonization represents two of the most rel­evant reactions for Csingle bondC coupling of biomass derivatives [15]. The former achieves effective and high-yield coupling between carbonyl — containing biomass intermediates at moderate reaction conditions. With regard to advanced biofuels, aldol condensation has been shown to be ef­fective for Csingle bondC coupling of biomass-derived furanics (contain­ing an aldehyde group) such as HMF and furfural with ketones such as acetone to produce diesel and jet fuel green hydrocarbon components [17], [18] and [19]. Ketonization, on the other hand, involves condensation of two molecules of carboxylic acid to produce a larger (2n — 1) symmetric ketone [20]. This reaction possesses a great potential for the catalytic up­grading of biomass since Csingle bondC coupling takes place with simul­taneous oxygen removal (i. e., the reaction involves the removal of CO2 and water) from carboxylic acids, the latter of which are common interme­diates in biomass conversion processes [21] and [22]. This reaction is typi­cally catalyzed by inorganic oxides such as CeO2, TiO2, Al2O3 and ZrO2 at moderate temperatures (300-425° C).

In the case of oligomerization and alkylation, they are especially indi­cated for upgrading of deoxygenated petroleum feedstocks (e. g. alkenes and hydrocarbons). However, relevant works by Dumesic and Corma’s groups have recently demonstrated that they can be successfully employed for advanced biofuels production in the last stages of biomass conversion processes, that is, when oxygen content in bio-derived feedstocks is very low [23] and [24]. Next sections will provide examples on the application of the abovementioned Csingle bondC coupling processes for the trans­formation of lactic acid and levulinic acid into advanced biofuels for the transportation sector.

The two main methods of infrastructure considered suitable for cultivation of algae are open (raceway) ponds or photo-bioreactors (PBRs) [55], and are compared in Table 3. Raceway ponds are similar to oxidation ditches used in wastewater treatment systems being large, open basins of shallow depth and a length at least several times greater than that of the width. Raceway ponds are typically constructed using a concrete shell lined with polyvinyl chloride (PVC) with dimensions ranging from 10 to 100 m in length and 1 to 10 m in width with a depth of 10 to 50 cm [55]. Oswald considered the open pond to be the most viable method of combining algal cultivation and wastewater treatment in the 1950s [56].

Photobioreactors are more commonly used for growing algae for high value commodities or for experimental work at a small scale. Recently, however, they have been considered for producing algal biomass on a large scale as they are capable of providing optimal conditions for the growth of the algae [55]. A closed reactor allows species to be protected from bacterial contamination, shallow tubing allows efficient light utili­sation, bubbling CO2 provides high efficiency carbon uptake and water loss is minimised. PBRs provide very high productivity rates compared to raceway ponds. In their life-cycle assessment (LCA) study, Jorquera et al. [55] estimated volumetric productivity to be at least eight times higher in flat-plate and tubular PBRs. The reason why PBRs however have not become widespread is due to the energy and cost intensity of production and operation. PBRs require a far higher surface area for the volume of algal broth compared to alternative infrastructure. Much higher volumes of material are therefore required which in turn requires a higher capital energy input and increases environmental impacts [57]. During operation algal biomass must be kept in motion to provide adequate mixing and light utilisation. These increase productivity but also require additional energy for pumping. So far in comparison to raceway ponds the benefits of PBRs do not outweigh the necessary energy requirements identified in the LCA study published by Jorquera et al. [55]. A net energy ratio (i. e., energy produced/energy consumed) of 8.34 has been reported for raceway ponds as compared to a net energy ratio of 4.51 and 0.20 for flat-plate and tubular photobioreactors, respectively [55]. It is likely that ponds will continue to provide the most effective infrastructure for algal cultivation due to their low impact design and low energy input requirement. PBRs will continue to be important however, for laboratory work, developing cultures and producing biomass with high economic value. As research continues it may also be possible to develop infrastructure that will provide the ben­efits of both PBRs and open ponds together.

We also included the sustainable share of current use of biomass for tradi­tional uses in the category ‘Complementary fellings’. This biomass is cur­rently primarily used for domestic heat production. We worked upon the assumption that the majority of this use is woody biomass, though other sources will contribute.

The demand side scenario, to which this work is related [1], postulates that any traditional use of biomass that is considered unsustainable today will be gradually phased out and replaced with more sustainable approach­es, such as solar thermal heating.

No literature data was available on the sustainable share of current traditional biomass use. Therefore, as the scenario gradually phases out traditional biomass use, we have estimated that 30% of the phased out biomass can be harvested sustainably. This amounts to approximately 11 EJ of worldwide potential for this category.

The biotechnology of fermentation using yeasts, like Saccharomyces cerevisiae, has a long history in many sectors of industry from alcoholic beverages to ethanol production. A vital focus of ongoing research is the study of the key enzymes responsible for the production of the metabo­lites of interest, namely ethanol. Increasing the activity of key enzymes, like alcohol-dehydrogenase, is a primary goal of metabolic and enzyme engineering. The glucose dehydrogenase and alcohol dehydrogenase were studied in S. cerevisiae under the influence of a non-uniform pulsed mag­netic field of 30 mT for 60 minutes [34]. They found that in the presence of NAD the glucose dehydrogenase activity increased 18%, while no effect was observed in the absence of NAD or NADP. The activity of alcohol dehydrogenase in the absence of co-enzymes rose to 10.7% in the an­aerobically cultivated cells and 19.9% in those cultivated aerobically. The activity of this enzyme increased by 20.5% when NAD was added to this enzyme in the aerobic culture, while an 8.5% decrease was observed in the anaerobic culture. Thus, the non-homogenous pulsed magnetic field of 30 mT stimulated the activity of the dehydrogenases, but behaved differently in the absence or presence of NAD and NADP.

The effects on ethanol fermentation by S. cerevisiae under the in­fluence of two styles of oscillating magnetic fields were studied by Perez et al. [35]. The primary magnetic field generator was composed of several permanent magnets stacked in series, while the recirculat­ing culture broth was directed through the intervening space of the magnetic fields where spatial orientation determined the desired inten­sity of 5-20 mT for each exposure. The recirculation velocity passing through the array of static magnets modulated the frequency. The sec­ondary generator was a double layer solenoid coil that produced 8 mT. Two magnetic field generators were coupled to the bioreactor, which were operated conveniently in simple or combined ways. The overall volumetric ethanol productivity enhanced by 17% over control at an optimum magnetic field treatment of 0.9-1.2 m s-1 velocity and 20 mT plus 8 mT solenoid. These results made it possible to verify the ef­fectiveness of the dynamic magnetic treatment since the fermentations with magnetic treatment reached their final stage, 2 h earlier than the control. Perez et al. [35] postulated that membrane permeability and the redox system that are affected by the electromagnetic field might have resulted in alterations of ion transport of the substrates. As a con­sequence, the cellular metabolism was stimulated for higher ethanol production.

Chloroform, ethanol, ethyl acetate, hexane, isopropanol, isooctane, metha­nol, and 2-ethoxyethanol (2-EE; Acros, Bridgewater, NJ, USA) were ob­tained from Fisher (Waltham, MA, USA) and were either HPLC or ACS reagent grade. Sulfuric acid and potassium hydroxide were reagent grade (Fisher). Sodium methoxide in methanol (0.5M), methyl oleate, methyl palmitate, di(ethylene glycol) vinyl ether (DEG), 2-dimethylamino meth­acrylate (DMA), divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), hexyl methacrylate (HMA), vinyl imidazole (IM), and azobi — sisobutyronitrile (AIBN) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Triolein was obtained from Nuchek Prep (Elysian, MN, USA) and mineral oil (Squibb) was purchased locally.

All HPLC solvents were degassed and filtered through 0.5 micron PTFE filters (Ominpore, Waters Corp, Milford, MA, USA) prior to use. Isooctane was dried by storing over calcium hydride and filtered before use. Amberlite® ion exchange resin CG-400 (100-200 mesh, chromato­graphic grade; Mallinckrodt, St. Louis, MO, USA) was prepared by wash­ing and settling in distilled water in order to remove fines, then dried at 55 °C.

As simple photosynthetic organisms, microalgae can fix CO2 and syn­thesize organic compounds, such as lipids, proteins and carbohydrates in large amounts over short periods of time. Traditional methods of microal­gae cultivation based on photoautotrophic mode have many shortcomings, among which low cell density is a major issue giving rise to low produc­tivity, harvesting difficulty, associated high costs, and hence poor tech­no-economic performance. Therefore, a signficant effort towards com­mercializing microalgae biomass production is to develop high density cultivation processes. Two approaches are being actively researched and developed: (1) metabolic pathways control; (2) cultivation system design.