Autotrophic CO2 fixation via the CBB cycle in R. eutropha is genetically determined by two operons (cbbLSXYEFPTZGKA), one of which is located on chromosome 2 (H16_B1383 to H16_B1396) and the other on megaplasmid pHG1 (PHG416 to PHG427). The chromosomal operon (cbbc) of the strain has a length of about

15.2 kilobase (kb) pairs comprising 13 genes. The second, highly homologous operon (cbbp) is located on the megaplasmid pHG1 and contains only 12 genes, totaling approximately 12.8 kb. With the exceptions of the triose-3-phosphate isomerase (tpiA, H16_A1047) and ribose-5-phosphate isomerase (rpiA, H16_ A2345), all enzymes of the CBB cycle are encoded in the cbb operons [52, 53]. The chromosomal and plasmid-borne cbb promoters in R. eutropha are functionally equivalent despite minor structural differences [54] . Transcription of all genes in either cbb operon depends on a single promoter upstream of cbbL [52, 54-56] that is subject to strong regulation [57]. Growth of R. eutropha under autotrophic condi­tions leads to high expression of the cbb operon genes [58]. The cbbR gene encodes for the transcriptional regulatory protein of the operon [55, 56]. In many organisms, the cbbR gene is typically located adjacent and in divergent orientation to its cognate operon. Inactivation or deletion of the cbbR prevents cbb operon transcription [55, 59]. The activating function of CbbR appears to be modulated by metabolites that signal the nutritional state of the cell to the cbb system. CbbR from R. eutropha is a sensor of the intracellular phosphoenol pyruvate (PEP) concentration. PEP increases the affinity of the activator to its operator target site, resulting in a decreased activat­ing potential of the CbbR protein in vitro [60] . This observation suggests that the role of CbbR in cbb operon transcription can thus be as an activator or a repressor.

The existence of subpromoters within the operons was excluded, and premature transcription termination thus represents an important mechanism leading to dif­ferential gene expression within the cbb operons of R. eutropha [61]. There is evi­dence for the participation of additional regulators in cbb control [59].

The enzymes of the entire CBB cycle are represented in Fig. 3. Additionally, the cbb operons contain cbbX and cbbY, which have no known function in R. eutropha. However, the gene rbcX from cyanobacteria, located between the two RuBisCO subunit genes, is responsible for the assembling the RuBisCO holoenzyme, together with chaperones GroEL/ES [62]. The CbbX protein product presents no homology to RbcX, but has conserved domains of the AAA family proteins, ATPase proteins
that often perform chaperone-like functions assisting in the assembly, operation, or disassembly of protein complexes [63].

The structure of the RuBisCO from R. eutropha was published (Protein Data Base 1BXN). As in most bacteria and higher plants with form I of RuBisCO, the enzyme complex is built up from eight large subunits and eight small subunits (L8S8) [64].

Distinct residues from each subunit of RuBisCO thus comprise the active site required for both carboxylation by CO2 and oxidation by O2, with the two gaseous substrates clearly competing for the same active site. The specificity factor (SF) is calculated as follows:

V K

CO2 O2

V K

O2 CO2

In (4), VCO, VO, KCO, KO refer to the Michaelis-Menten constants of

RuBisCO for the different substrates. SF defines the ratio between carboxylation and oxidation rates performed by each enzyme [65], but does not provide a direct measure of the rates, rate constants, or catalytic efficiencies of either carboxylation or oxygenation [66]. In R. eutropha SF = 75 [67].

The rate-limiting step in CO2 assimilation is catalyzed by RuBisCO, which is a very poor catalyst, exhibiting low affinity for CO2 and using O2 as an alternative substrate. Protein engineering could potentially be used to increase RuBisCO’s CO2 carboxylation activity, thereby increasing SF [68]. However, because both CO2 and O2 compete for the same active site [66], a short-term strategy to enhance IBT pro­duction would be to increase the intracellular CO2 concentration.

RuBisCO’s oxygenation reaction produces one molecule of 3-PGA and one mol­ecule of 2-phosphoglycolate [69]. The cbbZ gene, which encodes a phosphogyco — late phosphatase on the cbb operon might be an evolutionary adaptation in response to the presence of the 2-phosphoglycolate produced by RuBisCO during the oxida­tion reaction. CbbZ would prevent the accumulation of potentially toxic concentra­tions of 2-phosphoglycolate and rescue part of the carbon that would otherwise be lost through the glycolate metabolism [56]. In summary, the CBB cycle will play an important role in autotrophic IBT biosynthesis by R. eutropha and increasing car­bon flux through the CBB cycle is likely to enhance the IBT production rate.

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.

After the transesterification reaction, the post-processing steps needed to purify biodiesel according to the existing regulations and norms are the same as those involved in the biodiesel production from currently edible vegetable oils. Although the final product composition in terms of esters may be different depending on the feedstocks used, their physical properties are similar and no significant differences are expected between the two variants.

After the reaction is finished, the mixture is allowed to separate into an upper layer of methyl esters and a lower layer of glycerol diluted with methanol. Glycerol is removed by allowing the two phases to form and settle. Then, any unreacted alco­hol is air-stripped or vacuum-distilled away from the esters phase and recycled back to the reactor.

Depending on the process, water can be used to wash catalyst residues and sodium soaps from the methyl esters. Moreover, small amounts of concentrated phosphoric acid (H3PO4) can be added to the raw methyl esters to break down cata­lyst residues and sodium soaps. Predojevic [78] studied different puri fi cation steps of biodiesel obtained from waste frying oils, by a two-step alkali-catalyzed transesterification reaction, concluding that the best results are obtained when using silica gel and phosphoric acid treatments (with a yield of 92%) and the lowest yields (89%) are obtained using hot water. Also, Sabudak and Yildiz [83] applied three different purification methods to biodiesel produced from waste frying oils (water washing with distilled water, dry wash with addition of magnesol, and an ion — exchange resin) concluding that the most effective one is the ion-exchange resin.

The same situation occurs for the storage of biodiesel, where potential problems of decomposition may occur. For example, Lin and Lee [62] studied the oxidative stability of marine fish-oil biodiesel showing that the addition of antioxidant significantly retards the fuel deterioration over time, although it increases the kine­matic viscosity and carbon residue at the beginning of the storage period. These authors also concluded that the operating temperature is a dominant factor in the deterioration of the fuel characteristics.

Fig. 3 Energy consumption for stand-alone dewatering by centrifugation

TFF Disc stack Belt filter

■ HTR □ ELR

Fig. 5 Comparison of the energy consumption of dewatering units operating as a (a) stand-alone options and (b) options preceding flocculation

process by up to 98%. Flocculation is particularly critical when the culture volumes are extremely large. Heasman et al. [14] stated that a flocculation efficiency of 80% in a 24-h period is about the average standard requirement for a typical large-scale flocculation work. This study assumes no further processing requirement due to residual accumulation of flocculants, as shown by Lubian [18] that P. tricornutum achieved approximately 90% efficiency from diminutive flocculant dosages.

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].

Inflammatory processes are related with several cardiovascular diseases and oxidative stress, therefore its study is of high interest. Among anti-inflammatory compounds from algal sources astaxanthine, terpenes, sterols, indols, and shikimate- derivatives have been described [105] . There is a huge amount of enzymes and secondary metabolites involved in inflammatory processes, but the general trend is to measure the expression of some of those metabolites and/or enzymes when cells involved in the inflammatory response are “activated.” Leukocytes are among the most studied models; leukocyte migration has been shown to be one of the first steps in the initiation of an inflammatory/immune response and is essential for accumulation of active immune cells at sites of inflammation. The chemotaxis assay used to analyze the test material is designed to assess the ability of a test material to inhibit the migration of polymorphonuclear leukocytes (PMNs) toward a known chemotactic agent.

For example, polysaccharides from red microalga primarily inhibited the migration of PMNs toward a standard chemoattractant molecule and also partially blocked adhesion of PMNs to endothelial cells [101].

The specific organic constituents of brown algae are alginic acid, laminarin, mannitol, and fucoidan (Table 11). Among these compounds, mannitol lacks polymeric struc­ture; it is soluble, and can be easily transferred into the cell [139]. Mannitol can be utilized by anaerobic microorganisms with the formation of acetate and hydrogen as the major products, and minor production of ethanol, formate, lactate, and succinate [140]. The degradation of laminarin by anaerobic organisms was studied by inocula­tion of an anaerobic reactor with bacteria from the human gut [141]. The authors reported almost complete (>90%) usage of laminarin during 24 h with the formation of butyrate and other VFA. Alginate has a more complex molecular structure and usually forms a gel in algae. The alginate lyases are enzymes found to be responsible for alginate depolymerization [142, 143]. Biological degradation of soluble

Na-alginate gel is 6-8 times faster compared to Ca-alginate gel due to calcium cross bridging in the polysaccharides [144]. The products of alginate depolymerization are a mixture of oligosaccharides with different length [145], which are further degraded to 4-deoxy-L-erythro-5-hexoseulose uronic acid [146, 147]. The final products of alg­inate degradation are glyceraldehyde-3-phosphate and pyruvate [146, 147].

AD of fucoidan has not been studied in detail, but several fucoidan-degrading marine bacteria were isolated and characterized [148] . Several studies prove the possibility of AD of fucoidan containing waste sludge from alginate extraction [136, 149]. Some fucans are resistant to anaerobic fermentation possibly due to specificities of molecular structure in particular strains [148, 150, 151]. The AD of algal proteins and polyphenols and their impact on overall digestibility is an area that needs more attention. It is assumed that polyphenols associate with proteins and polysaccharides in the cell envelope that decreases their availability for biological degradation [150-152].

Brown algae are one of the most studied algal feedstocks for AD. Examined spe­cies include Macrocystis pyrifera, Ascophyllum nodosum, Durvillea antarctica, Sargassum spp., and Laminaria spp. According to the chemical composition, the theoretical methane yield of 0.52 L/g VS and 0.49 L/g VS were predicted for M. pyrifera and Laminaria sp. [79]. The authors reported an experimental methane yield for M. pyrifera of 0.43 L/g VS (82% VS reduction) but only 0.24-0.3 L/g VS (50-60% VS reduction) for Laminaria saccharina. The significant difference in VS reduction was explained by variability in chemical composition between these gen­era. Laminaria has a higher content of fucoidan, laminarin, and alginate but lower content of mannitol (Table 13) . The ratio between experimental and theoretical methane yield among M. pyrifera species is highly correlated with the mannitol content [153). Mannitol, in contrast to polysaccharides, can be easily and com­pletely degraded by anaerobic microorganisms (Fig. 8a) [79, 153]. The methane yield from L. saccharina samples harvested in spring (4.2% of mannitol and lami — naran, 23% of alginate from TS) provided only 50-65% of the methane yield in comparison to L. saccharina samples harvested in autumn (36% of mannitol and laminaran, 15% of alginate from TS) [154].

The methane yield from Sargassum spp. using a BMP test showed that two spe­cies from this genus Sargassum fluitans and Sargassumpteropleuron have small potential for biomethane production [121]. All tissues added at 0.12-0.20 L/g VS exhibited a methane yield that was only 33-46% of the theoretical methane yield (Table 18). Sargassum species appeared to be a poor feedstock for AD possibly due to low mannitol (3.5-4.5% from TS) and a higher content of fibers (36.5-40.6% from VS) (Table 13).

The ADP in continuously stirred-tank reactors proved that M. pyrifera is the best substrate for methane production among brown macroalgae tested. The digester fed by the M. pyrifera with higher mannitol content had large methane yield and stabil­ity at higher OLR (up to 9.6 gVS/L-day) in contrast to a digester fed with M. pyrifera with lower mannitol content or Sargassum species (Fig. 8b) . Using continuous reactors, the methane yield obtained for different species decreased in the following order: M. pyrifera (0.24-0.35 gVS/L-day), Laminaria sp. (0.2-0.28 gVS/L-day), D. antarctica (0.18 gVS/L-day), Sargassum sp. (0.08-0.15 gVS/L-day), A. nodosum (0.11 gVS/L-day).

The high cost of biofuel production and low efficiency of captured energy are major factors that limit the large-scale use of algae for biofuels. The integration of the ADP into the production of other high-value products (e. g., food supplements, phar­maceuticals, and clean water) from algae is likely to make AD economically attrac­tive for biofuel generation.

5.3.1 AD Integrated into Other Algal Biofuel Production Pathways

There are several algae to biofuel conversion technologies, such as lipids extraction followed by transesterification, thermochemical hydroprocessing, phototrophic microbial fuel cell (PMFC), and algal hydrogen production. These processes gener­ate large quantities of waste algal biomass, residues, or by-products. The ADP is a prospective technology that can convert these waste materials into valuable fuel.

Favorable oceanic deposits are those with a high SH in high-quality reservoirs. The challenges facing commercialization of marine GH are likely to be higher than those in the Arctic, given the higher cost deep water operations. Installed infrastructure and access to markets (e. g., in the GOM), or lack of an Arctic GH option (as is the case of India or Japan), may make this an attractive option. The oceanic deposits that serve as models for the evaluation of marine GH prospects are described below.

Offshore Japan— Nankai Trough. This area has probably experienced the largest investment and most advanced field research activity because of the intensive Japanese effort to evaluate the potential and feasibility of gas production from hydrates. Following the drilling of an exploration well in 2000 [190], a multi-well exploration program was conducted in 2004 at 16 locations in three different sites (Kumano Basin, Second Atsumi Knoll, and Offshore Tokai) that had been selected on the basis of the BSR signature [189] at water depths of 720-2,033 m (Fig. 8). A total of 32 wells were drilled, and a comprehensive evaluation was conducted. The experimental program focused heavily on the practicalities and challenges of well construction in hydrate sediments [189]. An offshore production test appears to be the next logical step [48,96, 172, 189].

МЕТІ “Tokai-oki to Kumano-nada”
exploratory test wells (2004)

Tokai-Oki

S>QQ2%D Seismic Survey Area

MITI “Nankal-Trough

2001 2D Seismic Survey Area

Water Depth 722m ~ 2033m Drilling Length 263m ~ 451m (LWD)

Fig. 8 The hydrate deposit areas in the Nankai Trough region offshore Japan, and the drilling sites of the 2004 Drilling Program [96]

GOM— Oligocene Frio Formation, Tigershark Deposit. Smith et al. [179] described this first documented case of high-SH hydrate-bearing sand in the Alaminos Canyon Block 818 of the GOM (Fig. 9). Log data from an exploration well in about 2,750 m of water at the site indicated the presence of an 18.25-m thick sandy hydrate-bearing layer (HBL) (3,210-3,228 m drilling depth) at a relatively high temperature (about 21°C), with a high porosity p (about 0.30), Darcy-range intrinsic permeability k, and with the base of the GH stability zone occurring at or slightly below the base of the hydrate [26,179].

This deposit belongs to the third tier in the resource pyramid of Fig. 2, with ini­tial SH estimates ranging from 0.6 to over 0.8 [26] . Preliminary simulations with synthetic data (describing GH reservoirs under the Tigershark conditions) indicate that such systems can reach gas production rates well in excess of 2.8 x 105 m3/ day= 10 MMSCFD [131,132].

GOM—GC955 and WR313 Deposits. The reservoirs are very recent findings that were first identified during the “Leg II” logging-while-drilling operations conducted in the deepwater GOM in April 2009 [11, 24, 142] by the GOM Gas Hydrates Joint Industry Project (JIP) [74,115,117,118]. The sites for Leg II drilling of the JIP were selected based on an integrated geologic-geophysical approach designed to identify accumulations of gas hydrate at high saturation SH (>50%) in sand reservoirs (Fig. 10).

AC818 Keathley Canyon

Fig. 10 Seafloor map of the Gulf of Mexico depicting the location of JIP Leg II drill sites: AC818, WR313, and GC955 [135]

The GC955 accumulation involves a sand-rich channel-levee complex at the mouth of the Green Canyon embayment. Two industry wells in the block have confirmed the presence of clean, thick sands in close proximity to the most clearly imaged channel axis. Logging results from Leg II showed a thick accumulation of gas hydrates within porous sand lithologies [54]. The drilling depth to the top of the sand unit was 389 mbsf (feet below sea floor). At the H-well location [54], approximately 100 m of GH-bearing media involved a sequence of thin horizontal sand-shale interbeds at approximately the 0.3-0.6-m scale. GH occurs in pore-filling mode in three separate zones with SH in the 50-85% range, with no indication of free gas.

The WR313 sites are within the highly dipping eastern margin of the Terrebonne salt-withdrawal mini-basin with significant sand deposition (see [115,117,118,177]). Two horizons were targeted for drilling in JIP Leg II: Well WR313 “G” and well WR313 “H” targeted the informally named “blue” and “orange” horizons, respec­tively. LWD data obtained at the G-well indicated a sand-rich interval with thinly interbedded sands and shales (0.3-1.5 m). The drilling depth to the top of the unit was 852 mbsf. About 12 m of GH-bearing sands within a gross interval of 21 m were identified, with SH typically ranging from 40% to over 70%. The primary con­trol on gas hydrate occurrence in this unit is availability of suitable reservoir condi­tions [9, 11] . The WR313 “H” horizon occurred approximately 140 m below the blue horizon, and consisted of two massive, clean sands with sharp bases and tops. Drilling depth to the top of the upper unit was 806 mbsf. The upper “orange” sand is 4 m thick, with SH as high as 90% [54]; the lower unit is about 7 m thick, with SH ranging between 35 and 80%.