Advanced gene manipulation tools are essential for an efficient application of genetic engineering technology, but they are still in the development stage. These gene manipulation tools include:

• Homologous gene replacement or nuclear gene targeting [346, 347]

• Inducible nuclear promoters [348]

• Gene silencing approaches [349-351]

• Gene expression regulation by riboswitches [352, 353]

• New protein tagging approaches [354]

Chloroplast Transformation Technologies

Commonly, wild-type green algae have heavily stacked thylacoids and large light­harvesting antenna complexes (LHC) acclimated for low light conditions that cause photoinhibition under high light conditions [355, 356]. To eliminate the formation of reactive oxygen species, the absorbed photons need to be released as fluorescence and waste heat. This release of energy reduces conversion efficiency of light energy to biomass [357]. One solution to increase the conversion efficiency is to reduce the LHC size and enhance light penetration to the growth media [357-360]. For exam­ple, RNA interference technology was applied to downregulate the entire LHC gene family in C. reinhardtii [361].

Metabolic Network Reconstruction and Simulation

A metabolic network model is a prospectively powerful tool for selection of the most suitable wild-type organism, as well as to provide direction for the genetic engineering of a more efficient mutant [362]. Combinations of knockout and added genes can be optimized for target product and/or biomass yield [363, 364]. A genome scale reconstructed metabolic network improved bioethanol production for Saccharomyces cerevisiae though genetic engineering and evolutionary adapta­tion [365, 366]. The optimal parameters of the designed bioprocess are the growth media composition, the level of irradiation, the temperature, the product yield, the physical dimensions, and the cost efficiency of the overall process [367] .

Knowledge of the occurrence of in situ GH is very incomplete, and is either based on limited direct evidence (hydrate samples) or inferred from other data. In perma­frost regions, direct evidence of gas hydrate is provided by ongoing R&D programs (discussed below), and by analysis of industry 3-D seismic data and data obtained during the drilling and logging of conventional oil and gas wells. In marine environ­ments, most of the inferences of GH occurrence are based on indirect indicators involving interpretation of relatively low-quality 2-D seismic data. Direct GH detec­tion and characterization from marine 3-D seismic data have recently been reported by Dai et al. [32]. The use of four-component ocean bottom seismic surveys has also shown great promise [4, 14].

Currently, the greatest potential for gas hydrate production is in units of sand lithol­ogy with high intrinsic permeability [10], a condition that (a) enables the fluid and gas migration necessary for gas hydrate has to accumulate to SH of 60% or more of pore volume, (b) allows easier transmission of destabilizing pressure and tempera­ture pulses from wellbores, and (c) provides the pathways by which dissociated gas can be produced. It is currently not well known how large the resource of gas hydrate that exists in sand reservoirs is, but it is clearly sizeable. Current best estimates are that the in-place resources within sand reservoirs in the GOM alone likely exceed 6,000 tcf of gas. Given expected recovery efficiencies, a technically recoverable resource exceeding 1,000 tcf or more is reasonable.

However, this sand-based resource is just the tip of the hydrate resource pyramid

[9] . Large volumes of in-place methane are known to exist within fine-grained sedi­ments in locations such as the Blake Ridge [148], offshore India [27], Malaysia [57], and Korea [176, 147]. Such occurrences may be relatively common, and may occur more widely than the sand-hosted variety. Economic and environmentally sound production from such deposits clearly faces enormous technical challenges, not only because of the leanness of the resource but also because of their adverse flow and geomechanical properties. While gas production from sandy HBS is con­ceivable using largely existing processes, it is clear that much more needs to be known, and perhaps fundamentally new approaches developed, to further the pros­pects of production from elements lower in the gas hydrates resource pyramid.

Pyruvate produced from the CBB cycle is converted to the key intermediate a-KIV by the BCAA biosynthetic pathway. AHAS catalyzes the first step in the biosynthe­sis of all three BCAAs (Fig. 4). It is capable of synthesizing (2S)-acetolactate, a precursor of valine and leucine from two molecules of pyruvate and synthesizing

(2S)-2-aceto-2-hydroxybutyrate, a precursor of isoleucine, from pyruvate and 2-keto-
butyrate. In most organisms, a single AHAS catalyzes both of the above-mentioned reactions, whereas in other organisms these reactions are catalyzed by separate enzymes [28]. The AHAS enzyme consists of two subunits, one being catalytic and one playing a regulatory role. No crystal structure with both subunits has been obtained to date, although individual subunits have been crystallized separately. Catalysis is believed to occur at the subunit interface, since the catalytic subunit alone has little to no activity [70]. Expression of AHAS is controlled by the amount of tRNABCAAs available in the cell. High levels of tRNABCAAs repress the transcription of AHAS. Additionally, AHAS activity is controlled allosterically at the activity level by its regulatory subunit, through binding of valine at the homodimer interface [71]. Combined site-directed mutagenesis and BCAA-binding studies have shown that valine binding at the homodimer interface potentially causes a conformational change resulting in a less stable complex with decreased enzymatic activity [72].

A promising approach to counter valine inhibition of AHAS can be found in

E. coli. E. coli has three AHAS isozymes, each with a different substrate specificity and regulation mechanism. The sequence of E. coli AHAS isozyme II differs from other AHAS, and its regulatory subunit is insensitive to direct feedback inhibition by valine [70]. R. eutropha AHAS (IlvBH in Fig. 1) shares most sequence similarity with E. coli AHAS isozyme III and is also subject to allosteric feedback inhibition by pathway intermediates (dihydroxyisovalerate and ketoisovalerate; Sinskey labo­ratory, unpublished data) and end products (valine, leucine and isoleucine; Sinskey laboratory, unpublished data). Thus, minimizing allosteric inhibition by products and intermediates is essential to optimize IBT production in R. eutropha. N-terminal amino acid residues that are conserved in all valine-sensitive AHAS could contrib­ute to the binding of valine or other BCAAs that cause allosteric feedback inhibi­tion. These residues can be mutated to the ones that are present in the valine-insensitive AHAS isozyme II from E. coli. C-terminal truncation studies on the valine-sensitive E. coli AHAS isozyme III show decreased valine inhibition. However, the exact mechanism of inhibition alleviation is unknown, since valine and other BCAAs are hypothesized to bind only at the N-terminus of the regulatory subunit [73] .

An AHAS enzyme’s selectivity (R) for aceto-2-hydroxybutyrate production over acetolactate production can be calculated by the following equation:

[AHB]/[2KB]

[AL]/[P]

where AHB, 2KB, AL, and P represent aceto-2-hydroxybutyrate, 2-ketobutyrate, acetolactate, and pyruvate, respectively. R values for E. coli AHAS isozymes I, II, III, and C. glutamicum AHAS are 2.0, 65, 40, and 20, respectively, all of which favor the formation of AHB over acetolactate. R. eutropha AHAS has R value of ~45 [28].

As actetolactate is a precursor of IBT, reducing the AHAS R value would help direct carbon flow towards a-KIV, and consequently IBT. Previous mutagenesis studies on the E. coli AHAS II catalytic subunit revealed a tenfold reduction in R when a tryptophan residue at position 464 was mutated to lysine, glutamine, or tyrosine [74]. It is suggested that the indole ring of tryptophan interacts with the
extra methyl group on 2-ketobutyrate and stabilizes it in the active site. Site-directed mutagenesis could also be used to decrease the R value for R. eutropha AH AS.

KARI, encoded by ilvC in R. eutropha, catalyzes the formation of 2,3- dihydroxyisovalerate from 2-acetolactate and the formation of 2,3-dihydroxy-3- methylvalerate from 2-aceto-2-hydroxybutyrate (Fig. 4). KARI has similar substrate preference towards both substrates. A unique feature of its reaction mechanism is that it simultaneously catalyzes both an isomerization and a reduction reaction. Mutations in active site residues that abolished the reductase activity also elimi­nated the isomerization reaction, suggesting that isomerization and reduction are coupled without any intermediate. KARI requires NADPH and a divalent metal ion, in most cases Mg2+, for catalysis. The metal cofactor is involved in the alkyl migra­tion isomerization step, whereas NADPH is the electron donor for the reduction step [28]. Since KARI has no substrate bias, simple overexpression of ilvC is likely sufficient to provide ample precursor amounts for the production of IBT.

DHAD catalyzes the formation of ketoacids from the products of KARI. The mechanism of action is unknown, but it likely involves the dehydration of vicinal diols to ketoacids via an enol intermediate. E. coli’s oxygen sensitive DHAD con­tains a [4Fe-4S]2+ cluster. The reaction mechanism is proposed to be similar to that of aconitase in the TCA cycle, which also involves an FeS cluster [28]. The activity, feedback inhibition and oxygen sensitivity of R. eutropha DHAD have not been studied. As shown in Figs. 1 and 4, the combined activities of DHAD, KARI, and AHAS convert pyruvate into the key IBT intermediate 2-KIV.

Robert Dillschneider and Clemens Posten

Abstract A variety of high value products have so far been produced with algae and the transition to algae mass cultures for the energy market currently arouses the interest of research and industry. The key to efficient cultivation of microalgae is the optimization of photobioreactors that does not only allow for efficient light cap­ture but also takes account of the specific physiological requirements of microalgae. Three fundamental reactor designs (bubble columns, flat plate reactors, and tubular reactors) are common and are discussed together with some elaborate derivatives in the following. Every concept excels with specific advantages in terms of light distri­bution, fluid dynamics, avoidance of gradients, and utilization of the intermittent light effect. However, the integration of all beneficial characteristics and simultane­ously the compliance with energetic and economic constraints still imposes demand­ing challenges on engineering.

As stated above, the conversion to biodiesel of waste oils and fats from the meat and fish processing industries represents an opportunity to valorize a residue and obtain a higher value product (biodiesel). In many situations, the adequate disposal of residual oils and fats represents an operational cost, as they cannot be burnt directly in a boiler without special equipment. Thus, from this point of view, there is an economic incentive to valorize those residues.

However, depending on the total quantity of residual oils and fats generated, dif­ferent approaches have to be considered. If the total amount is small, as it is the case of the waste frying oils generated in restaurants, it will be easier to make the selective collection of those materials to be processed in a centralized production facility. If a good logistic system is developed and properly implemented, and incentives are available for the residue producers, this is proved to be a good option [8], applicable even for the small — and medium-sized companies of the meat and fish processing industries. This situation may change if small and compact units for the production of small quantities of biodiesel from a wide variety of feedstocks become available, although the costs of energy and raw materials, and the hazards involved in the manipulation of dangerous chemicals may render this possibility impracticable.

However, if the quantity of residues generated is large, the option of having an in-house facility for the production of biodiesel may be viable from an economic point of view. In any case, the reduction in the consumption of fuel by the company, either by the utilization of biodiesel or by burning of the glycerol produced in the process, must be compared with the investment in equipment and operational costs due to the consumption of materials and energy necessary to produce biodiesel. With the increase in price of fossil fuels, this option is expected to become more and more attractive. It is also relevant for companies operating close by or even interconnected, that generate large quantities of residual fats, and that may be interested on a com­mon processing plant to take care of all the fat residues generated in their activities.

From an environmental point of view, the valorization of residual oils and fats to biodiesel production makes sense. This corresponds to the reutilization of a waste material originated from a renewable source, thus reducing the consumption of non­renewable fossil fuels. Although at a first glance there is a reduction in greenhouse gas emissions, in particular of carbon dioxide [71], the actual reduction depends on how the residues are collected and transported to the production site. To minimize those emissions, the logistical network should be properly optimized, for example, by giving the residue generators special containers for storing the waste fats and defining the more adequate collection routes. Although for in-house biodiesel pro­duction facilities this problem does not occur, additional savings may be accom­plished through an adequate process optimization and integration.

Moreover, with the advent of more stringent limits for greenhouse gas emissions and the development of trading schemes for carbon emissions, the production of biodiesel from residual oils and fats can be a good form to combine the environmen­tal and the economical aspects to one’s advantage. However, the current legislation and regulations still need to be improved or even created to be able to have a clear vision of the trade-offs involved on these decisions.

3 Conclusions

This article presents and discusses the main questions regarding the utilization of residual oils and fats for biodiesel production. Some key aspects are identified and strategies to deal with them are presented. Among them, the high content of FFA and moisture in waste frying oils and animal fats, as compared to fresh edible oils, makes the alkaline-catalyzed transesterification reaction to be less efficient for biodiesel production. A pre-treatment method suitable to handle this type of feedstocks is presented. Also, more efficient and robust production processes are presented that are able to use feedstocks with the characteristics normally encoun­tered in waste fats.

As the global demand for biodiesel increases and the pressure to be more envi­ronmental friendly, yet maintaining market competitiveness, increases, more and more waste residues will be seen as valuable raw materials. Besides helping compa­nies to fulfil their goals, policy targets defined at governmental and regional levels may be easier to reach.

Lipid extraction from microalgae can be performed in several ways. Some com­monly used technologies include supercritical fluid extraction, oil press extraction, solvent extraction and ultrasound-assisted extraction. Oil press extraction involves the use of machinery to literally squeeze cells until they rupture to liberate intracel­lular lipid contents. The types of oil presses used for extraction include the ram press, screw press and expeller press. Solvent extraction, which is the most com­monly used, involves the use of chemicals such as benzene, acetone and hexane. The interaction between the algal cells and the solvent causes cell wall rupture, thus causing equilibrial dissolution and liberation of intracellular lipids. Supercritical extraction makes use of fluid high pressures and temperatures (above the critical levels) to rupture the cells and liberate intracellular lipids. This method of extraction has proven to be time efficient but requires high operating cost [13]. The ultrasound technique makes use of cyclic sound pressure to rupture algal cells, and the resulting free intracellular lipid is harnessed using solvents. The advantages and disadvan­tages of each method are summarised in Table 4.

The extraction process involves the separation and purification of lipids from the dewatered biomass. The extraction technology investigated in this study is solvent extraction, involving lipid extraction as well as ethanol and lipid purification with a two-phase system of hexane and water. The only energy inputs for the extraction stage are the mixing and pumping requirements. Emissions from the mixing and pumping requirements are all Scope 2. The emissions due to mixing (1,127.28 tonnes of CO2-e/year) are significantly larger than the emissions due to pumping (87.53 tonnes of CO2-e/year). Mixing emissions account for 92.7% of the emission from the extraction phase due to large mixing and retention times during solvent extraction. The dewatered biomass volume and concentration are consistent for all three cultivation systems (HTR, ELR and RP), thus the emissions due to the extrac­tion of lipids from biomass generated from any of the cultivation systems are the same. This is 1,214.81 tonnes of CO2-e/year.

Chlorophyll is often used as a natural colouring agent due to its green colour. Its use in the food industries is becoming increasingly popular due recent legislation shifts which mandated the use of natural colouring agents in preference to artificial agents [51]. There are, however, disadvantages associated with use of chlorophyll as a colouring agent. Not only is chlorophyll generally more expensive than artificial colourings, but also tends to be unstable under the different pH conditions of the foods to which it is added. To resolve this instability, the chlorophyll molecule must undergo a chemical modification which replaces its magnesium centre with a copper ion before it is mixed with the food materials. Since the modified chloro­phyll cannot be metabolically absorbed and is eventually removed from the body as an excretion product, this complex is considered safe to replace the original

chlorophyll as a colouring agent in most developed countries. The concentration of free ionisable copper in the food must, however, be kept below 200 ppm under current regulations [20, 54].

It is well known that despite the bioactive (beneficial) compounds, several toxic compounds can be accumulated in algae and microalgae. Compounds like alka­loids, domoic acid, azaspiracid, brevetoxin, okadaic acid, pectenotoxin, or micro — cystins have been described.

Therefore, sometimes it is required to perform some toxicological tests mainly based in the mouse bioassay. Article 5 of a European Commission Decision dated 15 March 2002, laying down rules related to maximum permitted levels of certain biotoxins and methods of analysis for marine bivalve molluscs and other seafood states: “When the results of the analyses performed demonstrate discrepancies between the different methods, the mouse bioassay should be considered as the reference method.” The basic procedure involves i. p. injection of an extract of the sample containing the toxin and observing the symptoms. A deeper review on toxi­cological analysis can be read in the book edited by Gilbert and §enyuva “Bioactive compounds in Foods” [42] .