We have chosen three potential biofuel molecules as targets for which we will meta — bolically engineer R. eutropha (Fig. 4). We have targeted three classes of biofuel molecules: butanol, which will be derived from modifications to the PHB pathway; farnesene, which will be produced by introduction of a heterologous pathway to produce isoprenoids; and long-chain alkenes, which will be produced by modifying the fatty acid biosynthesis pathway and introducing genes for alkene biosynthesis. We have genetically modified wild-type R. eutropha to knock out the genes associ­ated with the PHB biosynthesis pathway [15]. We are comparing biofuel production from plasmid-encoded heterologous pathways in the wild-type strain with produc­tion in the PHB- strains.

Teresa M. Mata, Antonio A. Martins, and Nidia S. Caetano

Abstract The increased demand for biodiesel and the difficulties in obtaining enough quantities of raw materials for its production are stimulating the search for alternative feedstocks. Among the various possibilities, the utilization of residual fatty materials, in particular waste frying oils and animal fat residues from the meat and fish processing industries, are increasingly seen as viable options for biodiesel production. This work reviews the state of the art regarding the utilization of waste oils and animal fats as feedstocks for biodiesel production, which are characterized by the presence of high levels of impurities such as high acidity and moisture con­tent. The relative advantages and disadvantages of the different routes for biodiesel production are presented and discussed in this chapter, focusing on their chemical and technological aspects. Also discussed are the questions related to the viability and potential economic advantages of using this type of feedstocks in biodiesel production for road transportation.

T. M. Mata(H)

Laboratory for Process, Environmental and Energy Engineering (LEPAE), Faculty of Engineering, University of Porto (FEUP), R. Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal e-mail: tmata@fe. up. pt

A. A. Martins

Center for Transport Phenomena Studies (CEFT), Faculty of Engineering, University of Porto (FEUP), R. Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal

N. S. Caetano

Laboratory for Process, Environmental and Energy Engineering (LEPAE), Faculty of Engineering, University of Porto (FEUP),

R. Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal

Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic Institute of Porto (IPP), R. Dr. Antonio Bernardino de Almeida, s/n, 4200-072, Porto, Portugal

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_28, 671

© Springer Science+Business Media New York 2013

1 Introduction

As energy demands increase and the fossil fuel reserves are limited or are becoming harder and harder to explore, research is being directed towards the development of renewable fuels. This aspect is particularly relevant in the transportation sector, where the dependence on fossil fuels is even more evident and any possible alternative (e. g. fuel cells and hydrogen) is harder to develop and implement in practice. In the short term, especially in Europe, biodiesel (mono-alkyl esters of long-chain fatty acids) derived from renewable biological sources such as vegetable oils or animal fats are attracting a lot of attention. Among its main key features one can point out its renewability, biodegradability, improved viscosity, better quality of exhaust gases, and also the possibility of being used, as a petroleum diesel substitute or combined with diesel fuels, in conventional combustion ignition engines without significant modifications.

Biodiesel promises to supplement and even replace at a local/regional level fossil diesel while contributing to rural development and reducing the dependence on fos­sil fuels. However, under current production technology, its use in transportation even blended with diesel has some pros and cons. First, biodiesel production costs are higher than those of petroleum diesel, mainly due to its production from expen­sive edible vegetable oils that account for 88% of the total estimated cost for biod­iesel production [90]. This is one of the major hurdles in biodiesel commercialization, making it difficult to compete in price with fossil diesel and requiring in many cases subsidies or fixed prices policies to be competitive with current fossil fuels or to fulfil specific national or international targets for the incorporation of bio-based fuels. Second, the continued development, market growth, and market share of biodiesel, with the corresponding need of raw materials for its production, has risks of their own and is causing more harm than good. For example, some of the most relevant feedstocks, such as soybean oil and palm oil, are placing additional pres­sure on food supplies during a period of great demand increase in developing coun­tries and diverting valuable resources away from food production. Until new technologies and/or feedstocks unconnected with the human food supply chain are developed, the use of edible vegetable oils to produce biodiesel might further strain the already tight supplies of arable land and water all over the world, potentially pushing food prices up even further. Furthermore, biodiesel feedstocks are impacted by previous and current land use practices, and cultures are adapted to specific cli­mate and soil conditions available in restricted regions of the world. Thus, moving a culture from one region of the world to another will surely influence the crop yield potential. For example, requiring the utilization of more fertilizers, having an impact on the local biodiversity as some of the species can be invasive and displace native species, or bringing pests with them, with potential direct consequences to local ecosystems. Also, a more intense agriculture normally increases the soil erosion due to carbon loss and nitrate and phosphorous loss [82] .

To circumvent the problems referred above, new feedstocks are needed what is currently an extensive area of research. An example includes microalgae that have the ability to grow under harsher conditions, in areas unsuitable for agricultural purposes, and with reduced needs for nutrients. This way, the competition with other crops for arable soil, in particular for human consumption, is greatly reduced. Also, microalgae are easy to cultivate and can grow at low cost with little attention, using water unsuitable for human consumption. However, very high energy requirement for drying the algal biomass is a barrier to its commercialization at present [66] . Another example is Jatropha curcas L, currently at a very early stage of development for biodiesel production. Since the markets of the different products from this plant have not yet been properly explored or quantified, the optimum economic benefit of its production has not been achieved [57].

From the currently available alternative feedstocks for biodiesel production, some attention is being given to residual oil and fat, such as waste frying oils from restaurants or food industry, and animal fats resulting from the meat or fish process­ing industries, which otherwise need to be disposed off with care and represents an operational cost. Even though the residual oil or fat are of lesser quality than virgin vegetable oils and more difficult to process due to the presence of impurities or to their high acidity, they may be a good option for biodiesel production, allowing one to use a waste and treating it appropriately in the production of a product (biodiesel) with value that can be used internally by the company or sold out. Moreover, these fatty materials are available at a lower cost (in many cases even for free) and can be used as feedstock for biodiesel production. Araujo et al. [8] evaluated the biodiesel production from waste frying oil concluding that it can be economically feasible provided that logistics are well configured.

The most common animal fats that can be processed into biodiesel are beef tallow, pork lard, and poultry fat. Fish oils are also possible to be converted into biodiesel, although research in this area is not so advanced as for the animal fats. In most of these cases, the oil or fat are not readily available for use in biodiesel production, but need to be firstly extracted from the fatty residues. It is estimated that about 38% of the bovine, 20% of the pork, and 9% of the poultry are fatty material for rendering (e. g. bones, fat, head, other non edible materials, etc.) from which can be obtained about 12-15% of tallow, lard, or poultry fat that can be used for biodiesel production [25, 36].

The lipid content in fish varies a lot depending on the type of fish and by-product. For example, Gunasekera et al. [45] reported the lipid content of 17% in carp offal, 13% in carp roe, 57% in trout offal, 31% in fish frames, and 13% in “surimi” pro­cessing waste and fish meal. Oliveira and Bechtel [74] reported 11.5% of lipids in pink salmon heads and 4% in salmon viscera. Kotzamanis et al. [55] reported 12% of lipids in trout heads.

In the European Union, about one million tonnes of tallow is rendered each year

[69] . The United States generates in average about 4 kg/person of yellow grease per year, and based on this statistic, Canada should produce about 120,000 tonne/year of waste fats of various origins [100]. Brazil generates about 1,382,472 tonne/year of beef tallow and 194,876 tonne/year of lard from slaughterhouses, which is nor­mally used for producing meal and oil for animal feed [25] . The world fi sh capture and aquaculture production was in 2004 about 140 million tonnes of fish, from which about 25% was for non-food uses, in particular for the manufacture of fish oil and animal meal [ 34]. The amount of waste frying oil generated annually in several countries is also huge, accounting for more than 15 million tonnes, varying according to the amount of edible oil consumed. For example, the United States generates around 10 million tonnes of waste frying oils, followed by China with 4.5 million tonnes and by the European Union with a potential amount ranging from

0. 7 to 1.0 million tonnes [44]. However, the worldwide amount of waste oils generated should be much larger than that and it is expected to increase in the near future.

Some studies available in the open literature show some potential for these feed­stocks. For example, Chua et al. [30] performed a LCA to study the environmental performance of biodiesel derived from waste frying oils in comparison with low sul­phur diesel and concluded that biodiesel is superior in terms of global warming poten­tial, life cycle energy efficiency, and fossil energy ratio. Godiganur et al. [41] tested biodiesel from fish oil in compression ignition engines, showing overall good com­bustion properties and environmental benefits. In particular, there are no major devia­tions in diesel engine’s combustion and no significant changes in the engine performance. Moreover, there is a reduction of the main noxious emissions in com­parison with fossil diesel, with the exception on the nitrogen oxide (NOx) emissions. Wyatt et al. [97] produced biodiesel from lard, beef tallow, and chicken fat by alkali — catalyzed transesterification. The biofuel obtained from these animal fats were tested and the NOx emissions determined and compared with soybean biodiesel as 20% volume blends (B20) in petroleum diesel. Results show that the three animal fat-based B20 fuels have lower NOx emission levels (3.2-6.2%) than the soy-based B20 fuel.

Animal fats and vegetable oils differ on their physical and chemical properties. While vegetable oils have a large amount of unsaturated fatty acids, animal fats have in their composition a large amount of saturated fatty acids [20] . Animal fats such as tallow or lard are solid at room temperature. An exception is the poultry fat which is liquid at room temperature and has in its composition a low percentage of satu­rated triglycerides, comparable to soybean oil. Fish oils contain a wide range of fatty acids, some of them with more than 18 atoms in their carbon chain and even some with an odd number of carbons [37]. Chiou et al. [28] analysed and compared the methyl esters derived from salmon oil extracted from fish processing by-prod­ucts with methyl esters derived from corn oil, concluding that, although there are some differences in the fatty acid composition, salmon and corn oil methyl esters have similar physical properties.

The physical and chemical properties of waste frying oil and the corresponding fresh edible oil are almost identical, but differ from source to source depending on the oil source. Waste oils have normally higher moisture and free fatty acids (FFA) contents than fresh edible oil, particles of different composition, and also polymer­ized triglycerides are formed during frying due to the thermolytic, oxidative, and hydrolytic reactions that may occur [ 44] . Additionally, during frying, the oil is heated at temperatures of 160-200°C in the presence of air and light for a relatively long period of time, what contributes to increase its viscosity, specific heat, and darkens its colour.

For processing these fatty waste materials and to improve the quality of biodiesel produced, different solutions can be employed. For example, Guru et al. [46] stud­ied biodiesel production from waste animal fats in a two-step catalytic process and adding organic-based nickel and magnesium compounds as additives in order to achieve a reduction in the biodiesel pour point. Canoira et al. [22] evaluated biodiesel obtained from different mixtures of animal fat and soybean oil using a process simu­lation software (Aspen Plus™), concluding that a mix of 50% (v/v) of both raw materials is the most suitable to obtain a final product with a quality according to the standards and with the minimum costs. This is relevant to optimize the production processes and ensure that the costs of disposal should be higher than the costs of making biodiesel corrected by the potential economical gains, for example reducing the consumption of fossil fuels.

As most of the biodiesel feedstocks have similar characteristics, any improvement in the way how the pre-processing, reaction, and final processing are done, in particu­lar related to the reaction time and final product quality, will have a profound impact in the production capacity and in the overall process. As two phases are formed and the diverse reactants are presented in different phases, the effects of mixing are significant to the process. The interfacial area between phases increases with high mixing intensity, facilitating the mass transfer between phases and naturally increas­ing the reaction rates [10]. Noureddini and Zhu [73] confirm these conclusions and have shown that, depending on the reaction stage, both the mass transport and the reaction kinetics are dominant aspects controlling the process performance.

In this work, the various steps for biodiesel production are described, depending on the characteristics of the waste oil or animal fat, having in mind the process improvement.

The strain of microalgae considered for the design is P. tricornutum, which is a type of marine algae from the class Bacillariophyceae [31]. Figure 2 shows the proximate biochemical composition of P tricornutum. The design basis for the cultivation sys­tem is 50,000 tonnes of dry biomass per year. The cultivation system will operate 330 days per year with the number of batches dependent on the type of cultivation

system employed, as well as the specific yields and productivities. Seawater is used as the major source of water due to the marine nature of the algal specie. The life span of the plant is fixed at 10 years. It is assumed that 80% of the biomass is removed from the cultivation system at the end of each batch. After 330 days of cultivation, the facility will shutdown for major maintenance. Dominant strains of algae and unwanted parasites can often enter the reactor and destroy the culture [5]; thus it is pivotal that the cultivation system is shutdown for scheduled maintenance periodically. The cultivation system will rely on carbon dioxide from a power sta­tion, and this will be the only source of carbon dioxide for photosynthesis. The car­bon dioxide from the power station is assumed as a mixture with compressed air such that the mass fraction of carbon dioxide entering the cultivation system is 10%.

The major processes investigated as dewatering alternatives in this study include single-stage dewatering using centrifugation, chamber filtration, vacuum filtration, suction filtration and a dual-stage process using flocculation followed by centrifuga­tion. In comparing the dewatering of different cultivation options, the raceway pond was approximately 15 times more expensive to dewater using a single-stage process than the reactor-style systems. This is primarily due to a combination of the significantly larger volume of the raceway pond and its much lower concentration of biomass, which requires the dewatering equipment to run for a greater length of time to process comparable amounts of dry biomass, leading to exorbitant energy costs. The costs of dewatering biomass from the raceway pond cultivation system using the four options investigated can be seen in Fig. 11. The capital costs neces­sary in the dewatering stage were found to be very low, whereas the contribution of running costs was found to be significantly larger. Of the alternatives shown in Fig. 11, the chamber and suction filtration options were found to be significantly cheaper than the single and dual-stage centrifuge systems, chiefly due to their lower electricity consumption.

However, the large consumption of electricity demanded by single-stage centrifugal recovery has other major environmental and economic impacts, which makes the dual-stage dewatering process the preferred option. As previously noted, the costs in dewatering biomass from the reactor-style cultivation systems were considerably less than the cost in dewatering raceway pond culture. In Fig. 12 filtration again appeared to be the cheapest dewatering option; however, there were a number of aforementioned hidden costs associated with fouling which were not included in the model. Significantly, as shown by the dot shaded bar in Fig. 12, the higher capital costs relative to running costs made dual-stage dewatering uncom­petitive at this smaller reactor volume and higher concentration of algae. Thus, due to its greater reliability and cost effectiveness, a single-stage centrifugal dewatering process would be the optimal production selection in the dewatering of reactor-style cultivation systems.

A perennial problem with any LCA is identifying reliable and representative data sources. LCI data are available for many common raw materials (e. g., polyvinyl chloride) and manufacturing processes (e. g., extrusion), and, generally speaking, the more common a process, the better characterized it is from an LC perspective. Naturally, having multiple sources of data for a single process allows the user to evaluate the reliability of each source. In the production of algae, there are a large number of materials and processes that have been modeled from a life cycle stand­point that are quite useful. For example, reliable inventory data for a number of fertilizers, flocculants, and other industrial chemicals is readily available from a number of sources as highlighted in Table 3. Similarly, unit operations like pumping centrifugation can be easily modeled from first principles to derive energy use under conditions relevant to the specific process of interest [22].

As discussed earlier, current studies are somewhat limited by the fact that few full-scale algae-to-energy facilities are in operation. This makes it difficult to esti­mate the emissions from specific applications. For example, fugitive emissions from open ponds are expected to be nontrivial, and loss of this nutrient-rich medium could impact nearby receiving waters. Estimating this potential for eutrophication is highly speculative until actual ponds are in place from which data can be collected.

Similarly, there is little data to support assumptions about how often tubular photobioreactors would crack and require replacement, or the extent to which geotextiles are needed at the bottom of an open pond to prevent seepage of growth medium into the subsurface. Most of these estimates will be generally unreliable until some pilot plants are built in the coming years. In the meantime, analogous processes can sometimes be used to approximate the emissions associated with algae-related unit operations. For example, belt filter presses in wastewater treatment sludge handling can be used to approximate the impacts from an algae-to-energy unit operation, and as such, have been used by a number of authors [8, 31].

Miguel Herrero, Jose A. Mendiola, Merichel Plaza, and Elena Ibanez

Abstract At present, functional foods are seen as a good alternative to maintain or even improve human health, mainly for the well-known correlation between diet and health. This fact has brought about a great interest for seeking new bioactive products of natural origin to be used as functional ingredients, being, nowadays, one of the main areas of research in Food Science and Technology. Among the different sources that can be used to extract bioactives, algae have become one of the most promising. Algae have an enormous biodiversity and can be seen as natural factories for producing bioactive compounds since either by growing techniques or by genetic engineering approaches, they can improve their natural content of certain valuable compounds. In this book chapter, a revision about the different types of bioactives that have been described in algae is presented including compounds, such as lipids, carotenoids, proteins, phenolics, vitamins, polysaccharides, etc. Also, the modern green techniques used to achieve the selective extraction of such bioactives are presented and the methods for fast screening of bioactivity described.

Heterokonts are a large and very diverse group of algae identified relatively recently that possess similar morphology, photosynthetic pigments, ultrastructural features, and genetic code [50, 51] . Several Heterokont groups of algae correspond to for­merly distinct phylum, the Chrysophyta and Phaeophyta. The species are wide­spread in brackish water, freshwater, marine and terrestrial habitats. And chlorophyll a and c are the main photosynthetic pigments, and the main carotenoids are fucox — anthin or vaucheriaxanthin [52] (Tables 11 and 12).

Several Heterokont algae are used in mariculture. Isochrysis galbana, Chaetoceros muelleri, as well as diatoms Skeletonema costatum, Nitzschia and Navicula spp. are used to feed molluscs; Nannochloropsis is used to feed rotifers [53]. In the past

several decades, diatoms were studied for the production of pharmaceuticals, health products, biomolecules, nanomaterials, and bioremediators [54]. Several diatoms and Pinguiophyceae species have large biotechnological potential due to their high content of eicosapentenoic acid [55, 56] .

The brown algae (Phaeophyceae) are among the most cultivated algal species. Several brown algae were found to be good feedstock for AD. The composition of several species is presented in Table 13.

The alginate is a linear (1-4)-linked glycuronan composed of b-1,4-D-mannuronic acid and a-1,4-L-guluronic acid [57]. Mannitol is an alcohol.

Fucoidan structure varies in different algal species. Generally, it is a nonuni­formly branched and sulfated polysaccharide with backbone composed of alternat­ing 1-3- and 1-4-linked a-L-fucopyranosyl units [58, 59] (Table 14).

Genetic technologies make use of algae as a biological factory for the production of valuable algal metabolites and recombinant proteins [335] including:

• Carotenoids [336, 337]

• Long-chain polyunsaturated fatty acids [338]

• Pharmaceutically active compounds [339, 340]

• Polysaccharides [341, 342]

• Diagnostic and therapeutic recombinant proteins [343, 344]

Originally, genetic techniques were developed for three laboratory model organ­isms: C. reinhardtii, Volvox carteri, and P. tricornutum. Recently, genetic engineer­ing techniques have expanded to other algal species, including Chlorella sp. and diatoms. Sequenced genomes of algae are still limited. Green algae have only 3 genomes completed and 12 genomes are on assembly stage or in progress; diatoms have 2 completed genomes and 3 in progress. The process of sequencing 70 cyanobacterium genomes is completed and 102 genomes are on assembly stage or in progress [345] .

Gas can be produced from GH by inducing dissociation, which also releases large amounts of H2O (1). The three main methods of hydrate dissociation are (1) depres­surization, in which the pressure P is lowered to a level lower than the hydration pressure Pe at the prevailing temperature T, (2) thermal stimulation, in which T is raised above the hydration temperature Te at the prevailing P, and (3) the use of inhibitors (such as salts and alcohols), which shifts the Pe-Te equilibrium through competition for guest and host molecules [111] . Long-term production strategies often involve combinations of the three main dissociation methods [ 131, 132] . Another production method involves CH4 exchange with another hydrate-forming gas (e. g., CO2) through a thermodynamically favorable reaction [52, 213].

2 Occurrence, Research Activities and Priorities, and Prospective Production Targets

The geomechanical response of HBS in general, and potential well instability and casing deformation in particular, are serious concerns that need to be addressed and understood before gas production from hydrate deposits can be developed in ear­nest. Deposits that are suitable targets for production often involve poorly consoli­dated sediments that are usually characterized by limited shear strength. The dissociation of the solid hydrates (a strong cementing agent) can degrade the struc­tural strength of the HBS, which is further exacerbated by the evolution of expand­ing gas zone, progressive transfer of loads from the hydrate to the sediments, and subsidence. The problem is at its highest intensity in the vicinity of the wellbore where the largest changes are concentrated, and is further complicated by produc­tion-induced changes in the reservoir pressure and temperature. These can significantly alter the local stress and strain fields, with direct consequences on the wellbore stability, the flow and fluid properties of the system, the potential for coproduction of solid particles, and the overall gas production.

Recent coupled flow-geomechanical simulations investigated wellbore and res­ervoir instability during depressurization-based production from known oceanic and permafrost-related hydrate deposits [169-171]. The modeling results show that geomechanical responses during depressurization-based gas production are driven by the reservoir-wide pressure depletion, DP, which is in turn controlled by the production rate and pressure decline at the well. The depressurization of the reser­voir causes vertical compaction and stress changes, which in most cases will increase the shear stresses within the reservoir, which in turn can induce shear failure. The effect of pressure depletion on subsidence and stress during gas production from an oceanic Class 2 deposit in Fig. 16 shows that subsidence is proportional to the mag­nitude of DP, and depends on the elastoplastic properties of the HBS. In general, subsidence will be much larger in oceanic HBS because of much larger DP than in the case of permafrost-associated deposits. For the example in Fig. 16, the subsid­ence is about 2.5 m, and is a result of compaction in the hydrate-free, relatively soft, zone of mobile water. In the case of production from permafrost deposits at Mallik and Mt. Elbert, DP was limited to a few MPa, resulting in a subsidence of only a few centimeters and a compaction strain of less than 1% [171]. Subsidence in this case is also reduced as a result of a relatively stiff permafrost overburden. A general observation is that subsidence occurs uniformly over a large lateral distance from the well, and may thus be less of a hazard to overlying structures.

Rutqvist and Moridis [170] showed that the likelihood of inducing shear failure in the reservoir (a) depends on the initial stress field and the Poisson’s ratio of the host sediment, and (b) it is higher in the case of an oceanic HBS. If the stress field is initially near critical stress for shear failure, even a small pressure decline could suffice to trigger shear failure in parts of the dissociated reservoir, thus enhancing subsidence and sand production.

Stress changes and associated strain resulting from depressurization strongly affect well stability and the load on the well casing [168]. In vertical wells, the pres­sure depletion will generally unload the formation uniformly in a plane normal to the axis of the well, and the load on the well casing will decrease. In horizontal wells, vertical compaction of the formation acting against the upper part of the relatively stiff well casing will likely cause shear failure in the formation in that area. Such shearing of the formation can break the bonds between particles, resulting in sand production and creation of cavities around the wellbore. Several studies indicate the difficulty of avoiding shear failure in the formation around the production intervals of the wells, and highlight the need for appropriate engineering measures to prevent solid production [127].

HBS stress changes and the vertical compaction can be substantial in oceanic HBS. Moreover, formation failure may also occur in the form of pore collapse, in which the mean effective stresses increase so much that inelastic grains slippage and rearrange­ment occurs. Oceanic HBS are often at the highest effective stress in their geological life, which means that their pre-consolidation pressure would likely be exceeded during depressurization-induced production. Under pore collapse, f and k may be subjected to more substantial, and often irreversible, changes. Such processes and their affect on the gas production from the HBS need to be further investigated.