Co-digestion of different substrates has been recognized recently as an attractive approach that has many economical, logistic, and sanitary benefits [404]. The main advantage of co-digestion is the improvement of the nutrient balance that leads to greater reduction of VS and methane yield [405] . The C:N:P ratio is an important factor for AD as discussed earlier in the chapter. Co-digestion of low nitrogen bio­mass (municipal solid waste, paper, sisal pulp, straw, grasses, wood wastes) with higher nitrogen wastes (sewage sludge, chicken or livestock manure, slaughter­house, meat or fish processing wastes) can increase the loading rate and methane yield up to 60-100% [399, 406-409].

The C to N ratio varies for macro — and microalgae due to significant differences in biochemical composition. Carbohydrates are the main components of macroalgal

biomass with concentrations between 50 and 70% of dry weight. Microalgae usu­ally have a carbohydrate content in the range 10-20% while the protein content can reach 30-50% in some species. These differences in biochemical composition influence the C:N ratios and the strategies needed for co-digestion processes with macro — or microalgae.

In terms of global distribution, oceanic hydrates constitute about 99% of the total GH resource [178], so that a 1% error in the ocean approximations could encompass the entire permafrost hydrate reserves [178]. Kvenvolden [98] compiled 89 hydrate sites shown in Fig. 1 [178]. At those locations, hydrates were:

1. Recovered as samples (23 locations, of which 3 in the permafrost and 20 in ocean environments).

2. Inferred from (a) Bottom Simulating Reflector (BSR) geophysical signatures (63 locations), (b) decrease in pore water chlorinity (11 locations), well logs (5 loca­tions), and slumps/pockmarks (5 locations).

3. Interpreted from geologic settings (6 locations).

A measure of the dearth of direct knowledge on hydrates compares this mea­ger list, which represents the entirety of the database of natural hydrates, to the huge body of information on conventional and unconventional oil and gas reservoirs [ 209 ] .

Given their relative abundance, marine GH occurrences will likely be the pri­mary targets for future R&D activities. However, given the favorable economics of conducting long-term field programs in the Arctic (as opposed to the deep water), it is expected that arctic R&D activities will also continue. Two countries, the United States and Japan, are making considerable R&D investments in the Arctic, under the reasoning that the information gained on the behavior of gas hydrate­bearing sand reservoirs can be readily transferred to the study of marine resources at a later date.

Fig. 1 I (63), recovered (23), and potential (5) hydrate locations in the world [98]

Given the susceptibility of particular types of hydrate deposits to geomechanical changes, and the possibility for severe stability and well stability consequences, the ability to monitor geomechanical changes by geophysical means is particularly appealing. A modeling approach that allows for the coupled simulation of hydrate production and corresponding geophysical measurements is a useful tool for con­straining the interpretation of geophysical data collected during a production test and for designing appropriate geophysical surveys.

This approach was used to conduct a feasibility study for using VSP measure­ments to monitoring production from a submarine hydrate accumulation in the GOM [89]. The study indicated that, (a) for an incoming P-wave source, the most

reliable indicators of changing conditions in the HBL appear to be converted

S-waves transmitted through the HBL and recorded below it, and reflected P-waves and converted S-waves recorded above the HBL, and (b) the response was strongly dependent on the chosen rock physics models (Fig. 17). Future improvements to this approach should include linkage to coupled flow-geomechanical codes (e. g., [170]). In addition, other types of measurements that provide complementary but lower resolution information, such as electrical and electromagnetic data, should be evaluated.

3.1 Carbon Sinks

As mentioned in the pathway overview, removing alternative sinks of pathway intermediates would improve the IBT yield. However, when the removal of carbon sink creates auxotrophy, supplementation of the essential nutrient will drive up the production cost. Promoter regions of genes exhibiting low-level constitutive expres­sion during conditions permissive for IBT production have been identified based on microarray analysis of R. eutropha during nitrogen limitation (Brigham et al., man­uscript in preparation). These promoters will be used to maintain low expression levels of genes encoding essential metabolic reactions, thus preventing auxotrophy while limiting carbon flux to alternative sinks.

The 3-phosphoglycerate (3-PGA) generated in the CBB-cycle is converted to pyruvate via glycerate-2-phosphate and PEP. Since pyruvate is the only substrate of valine synthesis, control of its concentration is essential for high IBT yield. As a key intermediate in central carbon metabolism, pyruvate concentration is tightly regu­lated, for example through the PEP-pyruvate-oxaloacetate node [27, 85]. The high PHB accumulation during nutrient starvation suggests that the stringent response of R. eutropha keeps a tight control on any sink but PHB synthesis. This notion is confirmed by the excretion of pyruvate when the PHB synthesis operon is disrupted [35]. This control of pyruvate sinks can be used to optimize IBT production using a two-stage fermentation strategy (see Sect. 4.2).

pathway and their roles in biosyn­theses of other molecules. (a) Pyruvate and (b) 2-KIV. Shown in bold are enzymes that utilize these key intermediates as substrates

The final precursor of valine, 2-KIV, can be decarboxylated by a heterologous Kivd to yield isobutyraldehyde, the direct precursor of IBT. However, KIV is also a substrate of seven additional enzymes in R. eutropha (Fig. 6). To optimize the flux from KIV to IBT, alternate utilization of KIV needs to be minimized.

Of the seven enzymes representing KIV sinks, at least three are likely to be essential for survival of R. eutropha without auxotrophy. Isopropylmalate synthase (LeuA1, Fig. 6) is essential for leucine synthesis, BCAA aminotransferase (IlvE, Fig. 6) catalyzes the final step of valine synthesis, and 3-methyl-2-oxobutanoate
hydroxymethyltransferase (PanB, Fig. 6) catalyzes the formation of 2-dehydropan — tanoate, a precursor of Coenzyme A (CoA) [86]. Since use of an auxotrophic strain for IBT production would increase the cost of production, the promoter exchange strategy discussed previously could be used to minimize the activity of the above enzymes.

The remaining four enzymes representing KIV sinks can likely be deleted from the genome without auxotrophy, either because the enzyme is nonessential or has a redundant function. Leucine dehydrogenase (B0449, Fig. 6) and aminotransferase AlaT (Fig. 6) catalyze the conversion of KIV to valine and are thus redundant with BCAA aminotransferase [ 86] . Additionally, the second copy of isopropylmalate synthase (LeuA2, Fig. 6) will be removed from the genome to minimize the amount of protein available. Finally, 2-ketoisovalerate dehydrogenase (BkdAB, Fig. 6) is involved in valine degradation [86]. Minimizing degradation of valine will reduce the necessity to synthesize it from KIV; thus removing the genes encoding this enzyme from the genome offers two potential benefits.

Appropriate mixing is the basis for sufficient mass transfer in bioprocesses and simultaneously prevents cell sedimentation. On the reactor scale, homogenous con­ditions in terms of equal supply with all nutrients and CO2 is mainly determined by convection while on a cellular scale, mainly turbulent dispersion and diffusion influence mass transfer. Turbulences in the liquid phase reduce diffusion barriers around gas bubbles and therewith enhance not only carbon dioxide supply for pho­tosynthetically active cells but also oxygen removal [23, 34]. Stoichiometric CO2 demand of microalgae is strain-dependent and influenced by the physiological state as well as product formation (e. g., lipid accumulation) but can be considered to be in the range of 1.65 g/g biomass up to 3 g/g biomass for oil rich algae [26]. Low volumetric productivities of phototrophic cultures together with their related CO2 demand imply that the intensity of mass transfer is generally less problematic than in heterotrophic bacterial cultures (up to two magnitudes smaller). Nevertheless, significant gradients along the way of gas bubbles through the reactor can occur. This is mainly caused by the fact that the light path length and therewith depth of a reactor is limited (see above). Consequently, scale-up is restricted to extension in the two other dimensions. Reactor geometries are not inherently comparable between different conceptual design approaches. The occurrence of CO2 and O2 gradients is particularly significant for reactors with long distances between several aeration and degassing points like in tubular reactors [39].

Therefore, hydrodynamics have to be carefully considered to make sure that high local oxygen concentrations, and thus a shift towards photorespiration, are avoided. For some species, oxygen concentrations higher than 120% air saturation can already cause inhibition. High oxygen concentrations can also cause [30] photooxidative damage when algae cultures are exposed to intense sunlight at the same time [10].

Similarly, a balanced distribution of CO2 shall ensure that the local CO2 partial pres­sure does not drop below 0.1-0.2 kPa in any region of the photobioreactor (Fig. 5) [54].

Fig. 5 CFD simulation of a plate bioreactor (height 1 m, width 0.5 m, thickness 0.1 m). The red lines indicate trajectories of volume elements, representing the axial dispersion

If partial pressure drops below that level growth kinetics can be limited [15, 49]. Gradients of CO2 are also related to gradients of pH in the reactor since pH and CO2 concentration are interconnected by the chemical equilibrium of carbon dioxide, hydrogen carbonate, and carbonate [39]. These coherences are shown in Fig. 2 by the intersection of the key areas, “hydrodynamics” and “reaction.”

Mixing times of 100 s (at superficial gas velocities usually below 0.05 m/s) are not unusual for bubble column reactors. Upward liquid movement is induced by aeration; downward movement occurs close to the reactor wall. Axial dispersion coefficients are influenced by the superficial gas velocity and are typically in the range of 0.01-0.02 m2/s. Radial dispersion coefficients, however, are one to two orders of magnitudes lower than axial dispersion coefficients, yet gain special importance in photobioreactors [8]. With regard to the superimposed light field and considering the fact that photosynthesis occurs especially close to the reactor wall, where enough light is available, an equalized gas distribution at the edge of the col­umn is desirable. In addition, radial dispersion fundamentally determines the resi­dence time of cells in dark and illuminated volume elements. Radial dispersion coefficients (especially in volume elements close to the edge of the reactor) can be increased with higher superficial gas velocities, yet shear stress imposed on the cells and high costs of increased auxiliary energy input considerably limit aeration rates.

The interdependency between “hydrodynamics” and “light distribution” (Fig. 2) is further addressed in the following section.

The results presented in this study indicate that in an oxygen-free environment at a temperature between 250 and 550°C, the methylating reagent TMAH directly con­verts algae biomass into the fatty acid methyl esters found in biodiesel. While algae, high in lipid content and rapid in growth, are an ideal biomass for biodiesel produc­tion, we suggest that this direct conversion process can be used with various forms of other types of biomass. For example, soybeans have been treated with TMAH/ methanol and shown to give similar fatty acid methyl esters [15]. This process is also unique among biodiesel conversion technology in that the biomass introduced may be used wet, partially dried, or dried. Removing water from algal biomass is viewed as an important challenge to making conversion to biofuels feasible. The traditional transesterification process, in which sodium hydroxide is used to cata­lyze the reaction, requires the exclusion of water [12, 23]. There is also a require­ment to remove free fatty acids in the transesterification process, mainly because the free fatty acids are converted to fatty acids salts which do not undergo transesterification. Grasset et al. have shown that the TMAH process can convert free fatty acids to their methyl esters [13]. Thus, we expect that removal of free fatty acids from algal oils would not be necessary. This, combined with the fact that com­plete water removal would be unnecessary, suggests that one could streamline any commercial production of biodiesel using our proposed TMAH thermochemolysis methodology.

Although high temperatures of 450°C produce the highest yield of biodiesel, the ideal temperature for this TMAH process is 250°C or lower. Because the residual biomass char is not significantly altered, it is likely to be more useful as a fertilizer than would be a more highly charred product that one obtains at higher reaction temperatures. In addition, maintaining the reactor at a low temperature consumes less energy.

Acknowledgments We thank R. L. Cooper, T. A. Egerton, R. L. Hubbard, R. Mesfioui, and C. L. Wingreen for their help with sample collection and analysis. We thank all of those individuals from various departments at Old Dominion University (ODU) whose research has been invaluable towards this project, especially C. Burbage, A. Gordon, H. Marshall, and A. Stubbins. We also thank the College of Sciences Major Instrumentation Cluster (COSMIC), at ODU for the use of their NMR facility. This work is supported by the Virginia Coastal Energy Research Consortium (VCERC) and funded through the Commonwealth of Virginia.

A basis of 90% total lipid recovery is assumed for design calculations. Using the proximal composition in Fig. 3 , the annual yield of oil from P. tricornutum with 90% recovery is ~9,000 tonnes. However, this amount could reduce when the extract is purified and all unsaponifiable components are removed. The design is focussed on the use of solvent extraction on wet biomass with no cell lysis unit operation. Lee et al. [17] demonstrated that cell lysis before solvent extraction does not significantly affect lipid yield. This extraction method is employed due to its high lipid yields and minimal extraction steps. It also involves the use of low-solvent quantities. The total amount of saponifiable lipids extracted from P tricornutum is approximately 6.4% of biomass (dry weight) [27]. Therefore, the daily yield of purified lipid from the extraction is approximately 7.78 tonnes. Figures 6 and 7 show the product yields from the crude lipid extraction and purification processes.

Teresa M. Mata, Antonio A. Martins, Subhas K. Sikdar, Carlos A. V. Costa, and Nidia S. Caetano

Abstract This chapter describes how to perform a sustainability evaluation of microalgae biodiesel through its supply chain. A framework for selecting sustain­ability indicators that take into account all three dimensions of sustainability: eco­nomic, societal and environmental, is presented. Special attention is given to a useful definition of the boundary for the system and to the identification of the rel­evant impacts associated with the biodiesel supply chain stages. A set of sustain­ability indicators is proposed for quantitative sustainability assessment, based on the impacts deemed relevant for each supply chain stage. Some qualitative arguments [10] [11]

are also presented to support the evaluation. Although microalgae appear to be superior in some respects to other currently used feedstocks, the development of large-scale microalgae production systems still needs further research.

1 Introduction

It is commonly accepted that our dependence on fossil fuel and the gradual rise of greenhouse gas (GHG) in the atmosphere are intimately coupled. Several strategies are being devised and currently implemented in the transportation sector of the economy to stem this GHG rise. Examples of Government and business strategies alike include the development of alternative fuels, more efficient engines or trans­portation means, transportation networks better fitted to the societal and economic needs of evolving human societies, among others. In the short term, biofuels, such as biodiesel or bioethanol, are seen as viable options to partially fulfill the objec­tives of reducing the environmental impacts, in particular of global warming.

Biodiesel has some important advantages over other currently sought potential solutions. It can be produced from a wide range of vegetable oils from agricultural crops (e. g. rapeseed, soy, sunflower, palm oil, hemp, among many others) or even residual materials, in particular animal fats from the meat or fish processing indus­tries that are difficult to dispose of. The technology and know-how needed to pro­duce it efficiently is already available, and setting up a production facility is relatively easy. The real challenge here is to have access to enough raw materials to meet the current demand, without compromising sustainable development. Although source-to-wheel assessments indicate that the use of biofuels in vehicles yields benefits in terms of GHG and other pollutant emissions (e. g. sulfur and nitrous oxides) when compared to petroleum-based fuels, their impact on the biodiversity loss and competition for arable land can be deleterious if general sustainability cri­teria are not met. Also, the precise amount of saved CO2 emissions depends on the feedstocks used, the production processes, and on several other factors.

Thus, it is fundamental that the emerging biofuels sector is built on sound sustain­ability principles. In that regard, the European Commission recently put forward a broad set of sustainability criteria for biofuels in the Directive 2009/28/EC [3] for the promotion of renewable energy sources, which complement the targets already defined by the European Union concerning the utilization of biofuels. Some of the sustainability criteria in this new directive include that no raw material should be provided from undisturbed forests with important biodiversity, no land with carbon stock (wetlands or continuously forested areas) should be converted for biofuels pro­duction, the use of land for the production of biofuels must not be allowed to com­pete with the use of land for the production of food, a minimum of 35% GHG savings has to be attained, and also, societal considerations must be taken into account.

However, the increase in production and even the announced targets for biodiesel has raised some problems of its own. Nowadays, vegetable oils (edible or non-edible) and animal fats are the main feedstock for biodiesel production. As vegetable oils are also used for human consumption, the competition for arable land and the expected increase in food prices have become significant concerns. Additionally, it increases the biodiesel production costs, hindering its usage, even if the environ­mental impact of biodiesel is smaller than that of fossil fuels. Production processes may not be most adequate and optimized for the available feedstocks. Also, to fulfill the EU target of 10% from domestic sources, the actual feedstocks supply and the domestic arable land available in Europe are not enough [17] . Moreover, extensive monoculture plantation, the conversion of high conservation-value forests, and other critical habitats for cultivation of biodiesel feedstocks are unacceptable. These habi­tats and associated biological diversity can be lost forever, due to the cutting of existing forests and the utilization of ecologically important areas [ 14] . Also of concern are jeopardizing food supplies of people living in developing countries that still strongly depend on agriculture.

Therefore, new feedstocks are needed to complement the existing ones. Examples include the utilization of agricultural crops not used for human consumption, such as lignocellulosic materials and microalgae, among others, with higher biomass productivity when compared with the currently used feedstocks [18]. All options have their specific advantages and drawbacks that have to be taken into account when selecting adequate feedstocks. As the majority of production processes asso­ciated with alternative feedstocks are still under development, decisions concerning their development and practical implementation should be made considering all three dimensions of sustainability: economic, societal and environmental.

Among the potential feedstocks, microalgae are increasingly seen as a viable option for the production of biodiesel and even other types of biofuels. This work attempts to evaluate the relative sustainability of microalgae biodiesel when com­pared with the currently used fuels, and to identify the key advantages and problems associated with their use for biodiesel production.

Microalgae contain both chlorophyll a and chlorophyll b. Intracellular chlorophyll a content can vary from 0.0041 g/g dried microalgae (Synechococcus sp.) to 0.0185 g/g dried microalgae (Nannochloropsis gaditana) [53]. In green microalgae, the ratio of chlorophyll a/chlorophyll b ranges broadly from 0.64 to 5, in contrast to higher plants which have a narrower range from 1 to 1.4. The chlorophyll content and profile of a microalgal species continuously change depending on its life cycle and cultivation conditions (medium composition, nutrient availability, temperature, illumination intensity, ratio of light and dark cycle, aeration rate). Certain green microalgae, such as Chlamydomonas, Chlorella, and Scenedesmus sp., have mutants that can synthesize chlorophyll in the dark during heterotrophic growth [53] .

Figure 2 shows the downstream processing steps required to produce chlorophyll from microalgae, while Table 1 provides a list of different technologies currently

available for each step. After the microalgal culture is harvested from its cultivation system, it is concentrated in the dewatering step to yield a wet paste. Afterwards, the microalgal pellet undergoes a pre-treatment step for preparation towards chloro­phyll extraction. The chlorophylls are then extracted from cellular materials before being purified in a fractionation step [24].

The protein content in algae can be as high as 47% of the dry weight [35] , but these levels vary according to the season and the species. The protein content of brown algae is generally low (5-15% of the dry weight), whereas higher protein contents are recorded for green and red algae (10-30% of the dry weight). Except for brown algae U. pinnatifida which has a protein level between 11 and 24% (dry weight) [35]. Higher protein level were recorded for red algae, such as Porphyra tenera

(33-47% of dry mass) [35] or Palmariapalmata (8-35 of dry mass) [121]. These levels are comparable to those found in soybean.

There are studies about the variation of protein content of marine algae as a func­tion of the seasonal period [1, 39]. Higher protein levels were observed during the end of the winter period and spring whereas lower amounts were recorded during summer.

The in vivo digestibility of algal protein is not well documented, and available studies about their assimilation by humans have not provided conclusive results. However, several researchers have described a high rate of alga protein degradation in vitro by proteolytic enzymes. For instance, the relative digestibility of alkali — soluble proteins from P. tenera is higher than 70% [38] . On the other hand, some compounds limiting the digestibility of alga proteins, such as phenolic compounds or polysaccharides, have been described. Studies performed on brown algae show the strong inhibitory action of soluble fiber on in vitro pepsin activity and their negative effects on protein digestibility [59] .

Typical amino acid composition of different species of algae is outlined in Table 2 according to Dawczynski et al. [23] . The quality of food protein depends on its essential amino acids. These algae present high concentration of arginine, valine, leucine, lysine, threonine, isoleucine, glycine, and alanine, although the predomi­nant amino acids are glutamine and asparagine. Glutamine and asparagine exhibit interesting properties in flavor development, and glutamine is the main responsible in the taste sensation of “Umami.”

The concentration of essential amino acids, such as, threonine, valine, isoleucine, leucine, phenyl alanine, lysine, and methionine, are higher in U. pinnatifida than in Laminaria sp. U. pinnatifida has higher concentrations of Lysine that has Hizikia fusiforme and Laminaria sp. has higher concentrations of Cysteine than has

U. pinnatifida. Interestingly, taurine is not a typical component of traditional European food and taurine content represents a nutrient feature which is character­istic of red algae, such as Phorphyra sp. Taurine is detected at low concentrations in brown algae varieties.

In general, algae possess proteins that have a high nutritional value since they contained all the essential amino acids in significant amounts (see Table 2).

The organoleptic characteristic of algae are principally due to their free amino acid profile [126], which in turn depends on environmental factors in its culture grounds [44]. Generally, the free amino acid fraction of algae is mainly composed of alanine, aminobutyric acid, taurine, ornithine, citrulline, and hydroxyproline [89].

Other proteins present only in red and blue-green algae are phycobiliproteins (phycocyanin in blue-green algae, phycoerythrin in red algae), a group of protein involved in photosynthesis. Purified phycobiliproteins can have several uses, such as cosmetics, colorants in food, and fluorescent labels, in different analytical tech­niques [33, 138], These proteins are characterized by having a tetrapyrrolic pig­ment, called phycobilin, covalently attached to their structure. Important medical and pharmacological properties, such as hepatoprotective, anti-inflammatory, and antioxidant properties [9, 10, 156], have been described and are thought to be basi­cally related to the presence of phycobilin. Besides, phycobiliproteins might have an important role in different photodynamic therapies of various cancerous tumors and leukemia treatment [157] , Different works have been aimed to the selective extraction and analysis of the phycobiliproteins from algae, such as Herrero et al. [53] and Simo et al. [174], that identified the two subunits of each protein, namely allophycocyanin-a, allophycocyanin-b, c-phycocyanin-a, and c-phycocyanin-b, from S. platensis. In the red microalga Porphyridium spp., the red-colored pigment phycoerythrin [62, 195] has been described.