Moridis and Reagan [131] showed that depressurization-induced dissociation appears to be the most promising gas production strategy in Class 2 deposits. They proposed new well configurations to maximize production and alleviate a persistent problem of substantial secondary hydrate (or ice) formation in a narrow zone (r < 10 m) around the well. Using the properties and conditions representative of the Tigershark formation and producing at an initial constant mass rate of QM = 19.2 kg/s (=10,000 BPD), Moridis and Reagan [131] showed (Fig. 13) that (a) QM cannot be

Fig. 11 Gas production from a class 1 hydrate deposit. Left: evolution of (a) the rate of CH4 release from hydrate dissociation, (b) the rate of CH4 production at the well, and (c) the corresponding rate replenishment ratio over the 30-year production period. Right: evolution of (a) the cumulative CH4 volume released from hydrate dissociation, (b) the produced CH4 volume at the well, and (c) the corresponding volume replenishment ratio over the 30-year production period [129]

maintained constant during the production period (but has to decline), (b) the gas production rate is highly variable, (c) it is encumbered by a long initial lead time during which little gas is produced, but (d) it can reach levels as high as QP=4.8 x 105 m3/day (=17 MMSCFD), with an average gas production Qav over the 5,660-day period of simulation is about 2.2 x 105 m3/day (=7.8 MMSCFD). This study showed very high recovery from hydrate deposits, although economic and geomechanical considerations may limit total recovery. Similar results were obtained from the study of an oceanic Class 2 deposit in the Ulleung Basin of the Korean East Sea [136] and (b) a permafrost-associated deposit in the North slope [133, 134], leading to the observation that QP on the order of several MMSCFD is attainable in Class 2 deposits despite significant differences in reservoir tempera­ture, HBS thickness, and salinity.

The use of horizontal wells can substantially improve gas production from such deposits and reduce the initial period of low QP [137]. Conversely, Moridis and Kowalsky [128] determined that QP was too low to justify considering such accumula­tions as viable targets in the presence of permeable boundaries and/or with a deep WZ.

The Electrofuels concept pushes the boundaries not only of traditional biofuels but of industrial biotechnology writ large, through the first demonstration of chemo- lithoautotrophy as a means to enable carbon fixation and the production of reduced products, including liquid fuels. Chemolithoautotrophic organisms are prokaryotes, either bacteria or archaea, capable of deriving energy from the oxidation of various reduced inorganic compounds. These organisms use inorganic carbon (CO2 and its various hydrated forms) as their sole carbon source and are capable of growth and metabolism in the complete absence of either sunlight or reduced carbon [ 42] . Intriguingly, some chemolithoautotrophic bacteria have been shown to function as a biocathode, assimilating energy directly from electric current. Chemolithoautotrophs derive energy from the proton motive force generated by electron flow from a reduc — tant (e. g., H2, H2S, Fe2+) to an oxidant (e. g., O2, S, CO2). It is this energy flow that provides the driving force for the synthesis of both the ATP and reducing equiva­lents (NADH/NADPH) required for carbon fixation (Fig. 1c).

A large group of chemolithoautotrophs produce energy from the oxidation of molecular hydrogen:

2H2 + O2 ^ 2H2O AG° = -474 kJ (1)

Ralstonia eutropha is a relatively common soil microorganism capable of auto­trophic growth on hydrogen. Ralstonia relies on three different [NiFe]-containing hydrogenases capable of hydrogen oxidation in the presence of oxygen [7] :

1. Regulatory hydrogenase (RH): The RH senses the presence of hydrogen and initiates the expression of both membrane-bound and soluble hydrogenases.

2. Membrane-bound hydrogenase (MBH): Found on the outer-cytoplasmic mem­brane, the MBH shuttles electrons from hydrogen oxidation into the respiratory pathway, creating the proton gradient necessary to drive ADP phosphorylation.

3. Soluble hydrogenase (SH): The SH generates NADH reducing equivalents by the oxidation of hydrogen and electron transfer through two flavin mononucle­otides separated by a [Fe-S] cluster to NAD+.

While all three [NiFe]-containing hydrogenases are capable of hydrogen oxida­tion, each activity is coupled to a unique and essential cellular function required for chemolithoautotrophic growth.

Another chemoautolithotrophic bacteria of potential use for the production of liquid fuels is Acidithiobacillus ferrooxidans. This microorganism recovers energy from the oxidation of ferrous iron (Fe2+) in the presence of oxygen:

2Fe2 + + 0.5O2 + 2H+ ^ 2Fe3+ + H2O AG = -66 kJ(pH2) (2)

Acidithiobacillus is an acidophile, consistent with the increased stability of fer­rous iron at lower pH. Evidence suggests that Fe2+ is oxidized outside of the cell; electrons are thought to first enter the cell through a c-type cytochrome (Cyc2) in the outer membrane, and then pass through a series of periplasmic redox carriers to a cytochrome c oxidase, which in turn drives ATP synthesis [69].

Outer-membrane c-type cytochromes may interact with metals for energy pro­duction in other bacterial species, including Geobacter sulfurreducens. Intriguingly, recent evidence suggests that Geobacter protein filaments, sometimes termed microbial nanowires, are capable of conducting electrons over long distances inde­pendent of cytochrome proteins [33]. This exciting observation suggests that natu­rally occurring electron-conductive biofilms might be scaled to support biofuel production.

Carbon fixation is diversified in chemolithoautotrophs. All photosynthetic organ­isms fix carbon through the CBB cycle, which reduces carbon dioxide to glyceralde- hyde-3-phosphate (G-3-P); this intermediate is subsequently converted to five- and six-carbon sugars. The pathway requires six NADPH and 9 ATP to convert three equivalents of CO2 into G-3-P, with a maximum efficiency 67% of the thermody­namic limit. The key enzyme of the CBB cycle is ribulose-1,5-bisphosphate car­boxylase oxygenase (RuBisCO), a protein evolved for plant growth. While well suited for this purpose, RuBisCO possesses many attributes that limit its utility for the production of liquid fuels, in particular a low specific activity and a competitive reaction with oxygen [36] . In contrast, many chemoautolithotrophs use non-CBB pathways for carbon fixation. Two such pathways—the reductive acetyl-CoA, or Wood-Ljungdahl, cycle and the reductive citric acid, or Arnon-Buchanan, cycle— are more efficient than CBB, approaching the thermodynamic limit (Table 1) [28, 47]. Other non-CBB pathways, such as the recently elucidated 3-hydroxypropionate — 4-hydroxybutyrate cycles use bicarbonate as the source of inorganic carbon which may improve overall CO2 fixation kinetics relative to the CBB cycle. Further, unlike terrestrial plants that require carbon flux for complex structural carbohydrates, uni­cellular autotrophs use non-CBB cycle pathways to directly produce acetyl-CoA, the key intermediate for biofuel synthesis. Additionally, the 3-hydroxypropionate bicy­cle has no O2 sensitivity and does not catalyze competing O2 reactions, enabling oxidative phosphorylation for the generation of ATP. Lastly, several carbon-fixing enzymes show activities much greater than that of RuBisCO, although these enzymes have yet to be thoroughly investigated.

The Electrofuels program leverages chemolithoautotrophy and a variety of largely unexplored carbon fixation pathways as a potential platform for biofuels

Chemolithoautotrophic Platform Organisms

Clostridium E. coli Acidithiobacillus Nitrosomonas

Geobacter Pyrococcus Ralstonia Desulfobulbus

Shewanella Synechocystis Rhodobacter Mixed communities

Fig. 2 Electrofuels portfolio (MSW municipal solid waste)

production, and is built on the thesis that chemolithoautotrophic-engineered systems can assimilate energy and fix carbon more efficiently than can photosynthetic systems. Each Electrofuels project [1] aims to demonstrate three technology mod­ules (Fig. 2):

1. Energy assimilation: The efficient capture of the energy required to drive ATP synthesis and carbon fixation is a key feature of the Electrofuels program. Chemolithoautotrophic organisms capable of growth on a variety of reduced inorganic substrates are under consideration, including Ralstonia eutropha (H2), Acidithiobacillus thiooxidans (H2S), Nitrosomonas europaea (NH3), and Acidithiobacillus ferrooxidans (Fe2+). Electrotrophs—organisms capable of assimilating energy directly as electric current—that reduce carbon dioxide to acetate with nearly 100% Coulombic efficiency are also under evaluation [39]. The Electrofuels program also includes projects seeking to genetically engineer heterotrophic organisms such as Escherichia coli to function as chemolithoauto — trophs. Such projects include, for example, growth and production on electro­chemically produced formate.

2. Carbon fixation: Chemolithoautotrophs, in particular some archaea, evolved car­bon fixation pathways relevant to the unique geochemical niches they occupy (Table 1). To date, five pathways that use equivalent or fewer ATP than the CBB cycle have been discovered [5]. Such pathways confer a variety of benefits to chemolithoautotrophs, including avoidance of the requirement for large quantities of an inefficient RuBisCO. Further, non-CBB pathways directly convert CO2 to
acetyl-CoA, rather than G-3-P, an intermediate that directly feeds biofuel synthesis. Various Electrofuels projects are evaluating the characteristics of each of these pathways, as well as designed pathways not found in the natural world.

3. Biofuel synthesis: The Electrofuels program is designed to produce energy dense, infrastructure compatible liquids fuels, and specifies the production of fuel mol­ecules with an energy density equal to or greater than 32 MJ/kg. A variety of fuel molecules that can either be used directly as fuels, such as butanol, or hydrocar­bons that require further catalytic processing, such as alkanes, isooctane, and triterpenes, are the targets of this program. To produce such species in high yield, the Electrofuels program leverages recent advances in metabolic engineering and synthetic biology to direct carbon and energy fluxes to final fuel molecules.

I n summary, the Electrofuels program considers previously unexplored path­ways and organisms for the high efficiency conversion of solar energy and inor­ganic carbon to complex biofuels. Considering the available biochemical options and a rapidly growing toolbox for the genetic manipulation of previously intractable microorganisms, the program offers multiple options for the conversion of durable forms of energy to biofuels. In some respects, the components of a complete path­way (energy assimilation, carbon fixation, and fuel production) are “mix-and — match,” and the program virtually considers more approaches than those actually constructed.

я-Butanol has favorable properties as a gasoline blending agent and provides a valu­able target to validate R. eutropha as a host for synthetic biology [14]. The PHB synthesis pathway in R. eutropha proceeds through 3-hydroxybutyryl-CoA, which is also an intermediate in the я-butanol synthesis pathway. Our strategy will be to divert the flux from 3-hydroxybutyryl-CoA to PHB and to redirect it to я-butanol. Recent work has demonstrated that high titers of я-butanol can be produced in E. coli by choosing heterologous genes judiciously and maximizing reducing equiv­alents available for я-butanol production [2]. Therefore, a detailed understanding of the expression of heterologous genes for я-butanol production in R. eutropha and the metabolic flux in R. eutropha PHB — mutants will be essential in achieving high titers of я-butanol.

Although я-butanol can be used directly as a gasoline replacement due to its higher energy content and lower water solubility and corrosivity relative to ethanol, it would only address short-haul ground transportation, since it could not be used to power aircraft or long-range rail and trucks. Dehydration of butanol to butylenes (C4) and oligomerization affords C8, C12 and C16 olefins with some disproportion­ation to non-oligomer C9 , C10, C11, C13, C14, and C15 olefins. These olefins can undergo double-bond isomerization, skeletal isomerization, cyclization and/or aro — matization, forming isoalkenes, cycloalkenes, and/or aromatic products. Hydrogenation of this mixture may provide hydrocarbons suitable for use as jet fuels. We will transform the я-butanol obtained from H2/CO2 cultivation of engi­neered R. eutropha to these hydrocarbon mixtures and evaluate them as replacement for jet fuel. We are currently exploring novel catalysts for the dehydration and oli­gomerization of butanol to hydrocarbon mixtures that resemble jet fuel.

Processes for producing biodiesel from fatty waste materials should be similar to those from vegetable oils, the dominant feedstock according to the European point of view. Nevertheless, the special characteristics of these feedstocks, in particular their high content in FFA, moisture, and other contaminants, such as dirt and other chemi­cals that appear during processing and/or utilization of these fatty materials, requires that additional processing steps are employed, such as pre-treatment operations.

Currently, the process most widely used industrially for biodiesel production is the alkali-catalyzed transesterification of triglycerides, with low molecular weight alcohols and operated in batch mode. This process is more efficient and less corro­sive than the acid-catalyzed transesterification, the reaction is faster, and requires lower amount of catalyst to carry out the reaction, presenting only problems in the glycerol separation. Also, the alkaline catalysts (NaOH, KOH, NaOCH3 , etc.) are the most commonly preferred and are cheaper than the ones employed in the acid — catalyzed process (H2SO4 , HCl, etc.). However, the alkali-catalyzed process has several drawbacks, in particular it is very sensitive to the lipidic feedstock purity, mainly operating in batch mode and needing large reaction times to obtain a com­plete conversion of oil, and has complex biodiesel purification steps after the reac­tion. For waste fats, these factors have to be considered explicitly to ensure a proper conversion of the fats to biodiesel, meaning that in most cases pre-treatment steps

Fig.1

are required. Figure 1 shows a simplified process flowsheet for biodiesel production from waste oil or animal fat with high acidity by alkali-catalyzed transesterification preceded by a pre-treatment by esterification.

The sizes of both photobioreactors (HTR and ELR) and the raceway pond are esti­mated based on the capacity to produce 50,000 tonnes of dry weight biomass annu­ally. The volume and area of the horizontal photobioreactor were scaled up from the data presented by Chisti [6], the external loop bioreactor was scaled up from the design by Acien Fernandez et al. [1] , and the raceway pond was scaled up from the Outdoor Test Facility design in Roswell, NM [31]. Table 1 contains the annual production of biomass for each cultivation system and the biomass produced and harvested per batch.

1.5 Dewatering

Methods such as centrifugation, pressure filtration, vacuum filtration and tangential flow filtration (TFF) are used unaided to dewater microalgal biomass. This section explores the use of different dewatering methods either alone or as a preceding step to the aforementioned unit operations for microalgae dewatering.

The cost of the extraction and transesterification stages was based on one biomass production process technology, with the results shown in Fig. 13. The extraction stage was found to be significantly more expensive than the transesterification stage. The major contributors to the extraction costs were the large fixed capital costs and the cost of large quantities of solvents, respectively. The main components of the fixed capital costs were the costs of large mixing tanks and the pumping capacity required in filling and emptying the tanks. The small cost incurred during transesterification resulted from the significantly reduced volume of materials that required processing, with only 7.8 tonnes of saponifiable lipid estimated to pass to

■ Cultivation □ Dewatering О Extraction ■ Esterification

Fig. 13 Extraction and transesterification costs for algal lipids the transesterification stage daily from the original 151 tonnes of biomass processed in the extraction stage. The electricity costs for both processes were minor, as shown in Fig. 13.

1.7.2 Overall Production Costs

Overall, considering the economic outcome, a raceway pond coupled with a dual­stage dewatering process would be the preferred method to produce biodiesel. Considering biodiesel as the only saleable product, production costs were estimated as approximately $74/L of biodiesel. The calculations are based on the assumptions that glycerol is allowed to be sold, residue from the process also sold as animal feed and carbon credits received as discussed in Sect. 7. However, including these in the model only reduced biodiesel production costs to $72.60/L. With petroleum-based diesel currently retailing at ~ $1.10/L, though this analysis incurred a -50% error, biodiesel from microalgae still remains far too expensive, as compared with tradi­tional fuel.

Allocation refers to the broad category of assumptions that are needed to disaggre­gate highly interconnected industrial systems such that environmental impact can be assigned to specific processes. Since these decisions often introduce subjectivity into the analysis, the ISO (International Organization for Standards) standard for LCA effectively recommend that whenever possible, allocation decisions should be avoided [13]. Allocation questions arise often in LCA for processes that are multi­input (e. g., landfills), multi-output (e. g., oil refineries), or in which recycling occurs between processes (e. g., using coal fly ash from coal power production as a cement substitute) (Fig. 3). To study the LC of asphalt production, for example, it is possi­ble to collect refinery-wide emissions estimates, but a question will remain about how to assign these impacts to asphalt as opposed to the other outputs from the plant such as gasoline, diesel, lubricants, and so on. The emissions can be allocated based on estimates of relative mass flow rates or the relative economic value of the out­puts. Frequently, neither of these seems particularly satisfactory, because neither allocation rule has particular physical significance. What ISO recommends instead is to increase the level of detail of the model to tease out physical relationships between processes or products and specific environmental burdens. In the refinery

example, this would involve zooming in on the workings of the refinery to identify which specific unit operations are required for asphalt production and then only include those. The ISO standard acknowledges that allocation decisions are a major source of subjectivity in most LCAs.

In the life cycle modeling of algae-to-energy systems, there are several multi­input or multi-output processes that are likely to influence the environmental burden calculations. For example, when modeling the life cycle burdens of using an algae — derived fuel, it is likely that the fuel will be burned as a mixture with petroleum- based fuels. To account for the burdens assigned specifically to the algae content of the truck’s fuel will require allocation. Similarly, coproducts from algae cultivation have been widely discussed since there are significant life cycle (and economic) credits to be had for producing high-value by-products along with an algae-derived energy source. At present, there is no consensus on how to allocate the burdens of coproducts in algae production, in large part because the chemistry of these by­products ranges greatly. A proposed algae-to-energy facility might ferment a por­tion of the non-lipid fraction of the cells to produce ethanol and assign itself credit for this production. But should this facility receive credits for avoiding the produc­tion of corn ethanol at some other location? Another plant might produce high-value pharmaceutical additives. Until normative assumptions are developed in the field it is imperative that researchers are transparent about their assumptions.

The delivery of large volumes of CO2, a waste product from many industries, to algae cultivation facilities requires some allocation judgments, which can impact the results. Most industrial carbon dioxide in developed countries is a by-product of ammonia production. An ammonia plant can be modeled and the emissions quantified, but how much of the burden should be assigned to carbon dioxide vs. ammonia? An idealized plan produces about the same amount of both, but ammonia is the higher economic value product. Carbon dioxide is captured as a valuable by­product but without the ammonia, the plant would not exist. Some in the LC field argue that carbon dioxide should have burdens allocated to it using market price of the two commodities, even though this is an imperfect metric since prices change over time. Others suggest that the burdens should be allocated using a mass balance, but again here, this strategy neglects the fact that the facility exists to produce the more high-value product, ammonia.

The important economic, cultural, and scientific development of our society has strongly contributed to changes in life-style and food habits. For instance, highly caloric and unbalanced diets are commonly consumed in developing countries; this fact, together with a decrease in physical activity has raised the incidence of cardio­vascular diseases, diabetes, obesity, etc. [41]. If we also consider the increasing life expectancies, it is easy to realize that different solutions should be found to reduce the expected health costs in a near future.

M. Herrero • J. A. Mendiola • M. Plaza • E. Ibanez (*)

Institute of Food Science Research, CIAL (CSIC-UAM), Nicolas Cabrera 9, Campus Cantoblanco, 28049 Madrid, Spain e-mail: elena@ifi. csic. es

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_35, 833

© Springer Science+Business Media New York 2013

One of the possible solutions are the so called functional foods. The concept of functional food as a mean to protect consumer’s health was developed at the begin­ning of the 1980s in Japan, based on several scientific studies demonstrating the correlation between diet and a lower incidence of chronic diseases [3]. In 1993, the Ministry of Health and Welfare established a policy for “Foods for Specified Health Uses” (FOSHU) by which health claims of some selected functional foods were legally permitted and regulated [4] . In Europe, in the second half of the 1990s, a working group coordinated by the European Section of the International Life Science Institute (ILSI) and supported by the European Commission, was created to promote the action FUFOSE (Functional Food Science in Europe, IV Framework Program) to encourage the scientific study on functional foods. A definition of func­tional food as “the food that besides its nutritious effects, has a demonstrated benefit for one or more functions of the human organism, improving the state of health or well-being or reducing the risk of disease” [26] was established. In this definition, it is necessary to emphasize some new aspects: (a) the functional effect is different from the nutritious one; (b) the functional effect must be demonstrated satisfacto­rily; and (c) the benefit can consist in an improvement of a physiological function or in a reduction of risk of developing a pathological process. Besides, the func­tional foods need to be effective at the normal consumed doses and should have a presentation typical of a food product. At present, functional foods are regulated in the European Union by the guideline approved in December 2006 (Regulation (CE) 1924/2006 of the European Parliament and of the Council, December 20, 2006: nutrition and health claims made on foods). In this directive, the nutritional allega­tions and/or healthy properties of the new products are regulated, including their presentation, labeling, and promotion.

Considering this background, it is easy to understand the interest that functional foods have raised not only for consumers, but also for the food industry. Thus, we can consider that a new, enormous market for the food industry has been opened; as Sloan in 1999 already suggested: “foods for the not-so-healthy” [180].

But, how it is possible to convert a traditional food into a functional food? Again, there is not a single answer since many approaches can be used in order to improve the beneficial action of a certain food, ranging from more or less sophisticated bio­technological processes to several other processes to remove or increase the content of a specific compound. Many times, a functional food is obtained through the addi­tion of a component or a series of ingredients that either are not present in the analo­gous conventional food or are present at lower concentrations. These ingredients are called functional ingredients and are mainly micronutrients, such as w3 fatty acids, linoleic acids, phytosterols, soluble fiber (inulin and fructooligosaccharides, called prebiotics), probiotics (microorganisms able to improve the activity in the intestinal tract and the immune system), carotenoids, polyphenols, vitamins, etc., able to exert a specific healthy action into the organism [45, 179].

Algae can be found in nearly any aquatic and terrestrial habitat, showing a huge biodiversity and various morphologies ranging from phytoplankton species to large kelp [129]. Algae are photosynthetic organisms that possess reproductive simple structures; the number of algal species remains unknown although has been estimated at between one and ten million [112] and, as mentioned, can exist from unicellular microscopic organisms (microalgae) to multicellular of great size (macroalgae). For instance, microalgae use light energy and carbon dioxide with higher photosyn­thetic efficiency than plants for the production of biomass [7, 113] and have been suggested as a source of biofuel production, to purify wastewater [123, 134], to extract high added value foods and pharmaceutical products, or as food for aquacul­ture [182].

In fact, algae are organisms that live in complex habitats sometimes submitted to extreme conditions (changes of salinity, temperature, nutrients, UV-Vis irradia­tion), thus, they have to adapt rapidly to the new environmental conditions to sur­vive, producing a great variety of secondary (biologically active) metabolites, which cannot be found in other organisms [18]. Moreover, most of them are easy to culti­vate, they grow rapidly (for many of the species) and there exists the possibility of controlling the production of some bioactive compounds either by manipulating the cultivation conditions or by using more sophisticated genetic engineering approaches. Therefore, algae and microalgae can be considered as genuine natural reactors being, in some cases, a good alternative to chemical synthesis for certain com­pounds. Therefore, considering the enormous biodiversity of algae and the recent developments in genetic engineering, investigations related to the search of new biologically active compounds from algae can be seen as an almost unlimited field, being this group of organisms one of the most promising sources for new products. In this sense, previous reports have suggested both, micro — and macroalgae as a very interesting natural source of new compounds with biological activity that could be used as functional ingredients [141, 142].

Moreover, another important aspect to be considered is the development of appropriate, fast, cost-effective, and environmental-friendly extraction procedures able to isolate the compounds of interest from these natural sources. In this chapter, green extraction techniques, such as supercritical fluid extraction (SFE) and pres­surized liquid extraction (PLE) together with ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) are presented, and applications to algae bioactive’s extraction are discussed. 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. In this chap­ter, a short description of methods for fast screening of bioactivity (mainly antioxi­dant activity) is included, considering chemical and biological methods. Finally, future research trends and research needs for the attainment of bioactives from algae are critically commented.

AD is a complex biological process performed by a consortium of anaerobic bacteria and archaea. In this section, we provide a description of the ADP biochemistry and microbiology, the influence of environmental and physicochemical parameters on process performance, and the importance of biogas composition and its application.

1.1 Biochemistry and Microbiology of Anaerobic Digestion

Algal biomass consists of a mixture of organic and inorganic matter. The organic part is composed of complex polymeric macromolecules, such as proteins, polysac­charides, lipids, and nucleic acids. The polymers appear in particulate or colloidal form. The ADP converts organic matter to the final products (methane and carbon dioxide), new biomass, and inorganic residue. Several groups of microorganisms are involved in substrate transformation and the overall process comprises multiple stages with many intermediate products. Generally, the process can be simplified to four consecutive steps: (1) hydrolysis; (2) fermentation or acidogenesis; (3) aceto — genesis; and (4) methanogenesis.

The overall transformation can be described by six distinct biological processes as shown in Fig. 1 (modified from [60]):

1. Hydrolysis of colloid and particulate biopolymers to monomers.

2. Fermentation or acidogenesis of amino-acids and sugars to intermediary products (propionate, butyrate, lactate, ethanol, etc.), acetate, hydrogen, and formate.

3. b-oxidation of long-chain fatty acids and alcohol fermentation to volatile fatty acids (VFA) and hydrogen.

4. Anaerobic oxidation or acetogenesis of intermediary products, such as VFAs to acetate, carbon dioxide, and hydrogen. This reaction is performed by obligate and facultative hydrogen producing species.

Hydrogen 7 ( com/ronents

Fig. 1 Flow diagram of complex organic matter anaerobic digestion (modified from [60]), where (1) hydrolysis; (2) fermentation; (3) b-oxidation; (4) acetogenesis; (5) acetoclastic methanogens; (6) hydrogenophilic methanogens; (7) homoacetogenesis

5. Transformation of acetate into methane by acetoclastic methanogens.

6. Transformation of molecular hydrogen and carbon dioxide into methane by hydrogenophilic methanogens.

The same group of microorganisms that are primary fermenters performs the first three steps. These biological processes are sometimes referred to as acidogenesis or the acid-phase [61]. The other important biological processes in AD are:

• Conversion of a variety of monocarbon compounds (e. g., formate, methanol) to acetic acid. This reaction is carried out by homoacetogenic bacteria.

• Reduction of sulfur compounds to hydrogen sulfide by sulfur reducing bacteria.

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] .