Wild-type R. eutropha will accumulate up to 80% of its dry cell mass as the carbon storage compound PHB when it encounters nutrient stress [34] . During this strin­gent response, R. eutropha stops growth and slows down central metabolism (Brigham et al., manuscript in preparation) so that most of the intracellular carbon flux is redirected towards PHB synthesis.

Eliminating the PHB synthesis pathway from R. eutropha causes an overflow of the intermediate pyruvate during nutrient starvation, which is excreted to prevent toxicity [35]. Because pyruvate feeds the BCAA synthesis pathway used to generate IBT [4], nutrient starvation could be used to maximize carbon flux towards IBT.

2 R. eutropha IBT Production Pathway

2.1 Hydrogenase Enzymes

Hydrogenases are classified in three families, the [Fe]-hydrogenases, the [FeFe]- hydrogenases, and the [NiFe]-hydrogenases, based on their catalytic-site metal cofactors [21]. Only the NiFe hydrogenases are discussed here, as all three hydro­genases present in R. eutropha belong to this family [17]. Furthermore, the RH are not discussed since it does not have a role in the hydrogen metabolism of R. eutro­pha H16 [22]. As mentioned previously, the two types of energy conserving hydro — genases, MBH and SH, produce the energy and reducing equivalents supporting autotrophic growth of R. eutropha. Because of their oxygen tolerance, these hydro­genases in R. eutropha have been studied in detail (reviewed in [17, 36]).

MBH uses extracellular hydrogen to provide reducing equivalents to the respira­tory chain, allowing the four-electron reduction of O2 to H2O. The electrons gained in oxidation of hydrogen at the NiFe catalytic site are transported through the three iron-sulfur (FeS) clusters of the electron transfer subunit to a cytochrome b in the membrane anchor [37]. From cytochrome b the electrons are directed into the qui — none pool. The proximal FeS cluster, closest to the catalytic site, is critical for oxy­gen tolerance of the MBH, which is inferred from rapid reduction of O2 bound to the catalytic site [38]. The required electrons for the three-electron reduction of the peroxide radical, bound to the active site after oxidation by O2, are transferred from the quinone pool.

The cytoplasmic SH uses intracellular H2 to directly reduce NAD+ to NADH, through electron transfer from its catalytic subunit, through an FeS-containing elec­tron transfer subunit, to a flavin containing diaphorase moiety consisting of two subunits [19]. The remaining two subunits create a binding pocket for NADPH, which is required for catalytic activation of the enzyme [39]. The autotrophic growth rate of R. eutropha decreases twofold in an SH deletion mutant, illustrating that it is necessary to support the high reducing equivalent requirement of the CBB cycle [20]. The oxygen tolerance of the SH is thought to have a different molecular basis than that of the MBH, since it cannot access the quinone pool as rapidly. The coor­dination sphere of the NiFe active site is proposed to contain two additional cyanide (CN) ligands, one coordinating each metal atom [40, 41]. However, a recent study suggests that these two CN ligands are not present in situ, suggesting an alternative, yet to be elucidated, mechanism for oxygen tolerance [42] .

In summary, both types of energy-conserving hydrogenase are oxygen tolerant at ambient concentrations and serve a distinct purpose in maintaining the energy bal­ance of the cell. Optimal IBT production requires a balance of both hydrogenase activities.

In order to obtain high-purity bioethanol, solids and other aqueous components associated with the bioethanol need to be removed by clarification and distillation respectively. This separation process, however, has not yet been demonstrated for microalgal-bioethanol broth. The residual biomass produced after the separation process can theoretically be concentrated and converted to other products, such as animal feeds or fertilizers. The purity of bioethanol must satisfy international stan­dards for fuel specifications, ASTM D5798—09B. The produced bioethanol can be either blended with gasoline to form E10 (10% bioethanol) and E85 (85% bioetha­nol) or used directly in vehicles as a substitute for gasoline. Each of the blends has its own specifications which vary from one country to another. The overall cost of bioethanol production from microalgae should be made low enough to compete with existing commercial fuels. Due to the lack of any existing pilot-scale produc­tion facility of bioethanol from microalgae, practical information on operating and production costs is not readily available.

Acknowledgement This work was supported by an Australian Research Council (ARC) Linkage grant between Bio-Fuel Pty Ltd (Victoria, Australia) and Monash University Department of Chemical Engineering (Victoria, Australia).

Microwave-assisted transesterification is another possibility for biodiesel produc­tion from lipidic feedstocks with high acidity [61]. Refaat et al. [80] compared both microwave-assisted and the conventional process for producing biodiesel from high acidity feedstocks, concluding that reaction time is reduced by about 97% and sepa­ration time by about 94% using microwave irradiation.

Perin et al. [76] compared the acid-catalyzed and alkali-catalyzed trans­esterification assisted by microwave irradiation concluding that the best results are obtained under basic conditions, i. e. the reaction takes place in 5 min, and 95% conversion is obtained. Azcan and Danisman [12] have considered microwave heat­ing to perform the transesterification of rapeseed oil, showing that increased yields and reduced reaction times are possible.

For design purposes, a basis of 50% weight by volume (w/v) of biomass is used as the minimum requirement from the dewatering stage. The design outlines the best dewatering configuration in terms of energy consumption and economics. It is assumed that the initial cultivation within the bioreactors will be staggered so that dewatering equipment costs can be reduced. The staggering of the cultivation is entirely dependent on the bioreactor configuration and the dilution rate employed.

The boundary for each system includes a flue gas pre-treatment phase which is com­mon for all three cultivation options. The flue gas pre-treatment phase involves the pumping requirement to blend CO2 with compressed air. The majority of emissions are Scope 2 electricity emissions, as shown in Fig. 14. The primary source of electricity in the chosen location is brown coal, and this is regarded as a high-emission intensive generator, thus the high emission factor of 1.22 CO2-e/kWh is used (NGER, 2008).

As seen in Fig. 14, Scope 2 emissions for the HTR and ELR (primarily due to electricity consumption by the airlifts) are significantly higher than the Scope 2 emissions for the RP (due to the low electricity consumption by the paddle wheel). The Scope 2 emissions for the HTR and ELR are 186,691.52 tonnes CO2-e/year and 166,916.41 tonnes CO2-e/year, respectively; whilst for the RP the emissions are only 10,564.07 tonnes CO2-e/year. Scope 1 emissions are due to the capturing efficiencies that exist in the cultivation system. For the HTR and ELP, the efficiency of absorption was set at 95% whilst for the RP the capturing efficiency was 90% [6]. Considering both emissions, it is clear that the RP is the best process option as it captures the highest amount of CO2-e (71,213.70 tonnes CO2-e/year). Note that in considering Scope 2 emissions, the audit considers emission produced in the gen­eration of electricity—an emission from another independent facility. Thus RP is the most “truly” environmentally friendly option, as it considers all possible GHG emissions due to the process. However, under the CPRS requirements, only Scope 1 emissions are considered. If the CPRS requirements are considered then the HTR and ELR capture the largest amount of CO — gases: 87,157.89 tonnes CO — — e/year. Thus if the algae cultivation process was part of an emission-intensive industry, the HTR and ELR would be the best option. However, it is important to consider eco­nomic factors such as the cost of electricity usage by the HTR and ELR systems.

Nutrient feeding during a fed-batch process can be done utilizing either constant or variable mass flow rate. Additionally, the addition regime can be either intermittent or continuous [14]. Pulse feeding is easier and less expensive because of the absence of pumping costs. On the other hand, it could lead to lower cell growth, productivity and nitrogen-to-cell conversion factor. During the continuous addition, a feed pump is always used for the continuous addition of substrate during cell growth. With intermittent addition, the substrate is added by pulses and the time between two pulses is another variable to be studied.

Danesi et al. [33] have evaluated the S. platensis growth using urea as a nitrogen source in a fed-batch process, and they tested different protocols for urea addition, namely:

1. Intermittent—exponentially increasing the amount added, every 24 h

2. Intermittent—exponentially increasing the amount added, every 48 h

3. Continuous—exponentially increasing the mass flow rate

4. Continuous—constant mass fl ow rate, using a controlled fl ow peristaltic pump

The addition of urea at intervals of 48 h led to the lowest maximum cell concentra­tion, as a consequence of the lack of availability of the nitrogen to the microorganism between feeding times due to the lost of the nitrogen source in the ammonia form.

The best results, in terms of cell growth, show that the continuous form (protocol (iii)) is appropriate, bringing about better use of the nitrogen source and increasing biomass growth. It was possible to achieve a gain of 37% in biomass and consequently larger total amounts of chlorophyll at a lower cost when compared with cultures grown with KNO3. The fact that the water is being continually replaced (and not just once a day) in such experiment is also favorable, because it avoids the build up of high salin­ity conditions in the culture medium, that can hinder cellular growth. Nevertheless, comparing the results obtained at three temperatures studied (27, 30, and 33°C), the average maximum cell concentration was only 5.7% higher when urea fed continu­ously instead of intermittent addition every 24 h.

On the other hand, Sanchez-Luna et al. [86] compared the influence of the proto­col of urea addition (pulse or continuous), under variable conditions of temperature and total feeding time, in order to select the best feeding regime for S. platensis fed — batch cultivation with constant flow rate. Urea was added to the culture by a fed-batch process at constant mass flow rate, following two different protocols: (a) intermittent addition every 24 h and (b) continuous feeding. Fed-batch cultiva­tion of this cyanobacterium with constant urea feeding rate by pulse and continuous additions exhibited statistically coincident results. Because of the large solubility of this nitrogen source in water, it could be intermittently added avoiding the use of pumping equipments. Therefore, the addition of urea by daily pulse feeding at con­stant flow rates could be a useful protocol to be used in large-scale aquaculture facilities, implying lower costs for A. platensis biomass production.

An exponentially increasing feeding rate to supply urea is suitable for those cul­tures in which microbial growth can be inhibited by this nutrient or its derivatives. In fact, the highest nutrient supply takes place just at the end of the run, when bio­mass concentration achieves its maximum value. In this way, despite the inhibitory effect of ammonia coming from urea hydrolysis under alkaline conditions, the use of such a nitrogen source allowed S. platensis to reach a concentration comparable with that obtained with potassium nitrate in a batch run [33]. On the other hand, Sanchez-Luna et al. [87] observed that fed-batch autotrophic A. platensis cultiva­tions, carried out at 6.0 klux (72 pmol photons m-2 s-1) in 5.0-L open tanks at vari­able pH, temperature, and urea molar flow rate (K) resulted in a cell growth that followed a linear trend likely due to light limitation [13], thus justifying the use of constant mass flow rates. The use of urea as a nitrogen source prevented the inhibi­tory effect observed with ammonium chloride, and the growth curves did not exhibit any lag phase. The yield of biomass based on nitrogen progressively decreased with increasing urea molar flow rate. The statistical model pointed out pH = 9.5, T= 29°C, and K = 0.551 mM d-1 as the best conditions optimizing cell concentration and pro­ductivity, which do not differ much from the experimental observations.

Ammonium sulfate and urea have been tested as nitrogen sources for S. platensis cultivations, according to different batch and fed-batch protocols. The results showed that the use of urea in fed-batch culture led to better growth kinetics. Adoption of an appropriate slowly increasing urea feeding rate prevented the accumulation of ammonia in the medium as well as its well-known inhibition of biomass growth [97]. Preliminary batch cultivations were carried out to determine the actual nitrogen requirements of biomass as well as to establish the inhibition threshold of ammonia. Subsequent fed-batch runs, performed according to different feeding protocols, allowed the selection of the best conditions for urea supply and demonstrated that growth kinetics may be comparable and even better than that obtained with the tra­ditional nitrate-based culture media. Ammonia accumulation that usually inhibits biomass growth was in fact prevented following an appropriate pulse-feeding pat­tern. Although the highest productivity during the start-up was obtained with linearly increasing feeding rate, the use of a more slowly increasing pattern, aimed at mini­mizing ammonia accumulation, was shown to be the most suited for long-term culti­vation. Therefore, the use of urea in a fed-batch process should be recognized as a possible way to decrease the costs of a large-scale plant for the production of this cyanobacterium.

In a recent study, Ferreira et al. [41] cultivating A. platensis in a tubular photo­bioreactor, which is useful in preventing ammonia loss by evaporation, observed that parabolic protocol for ammonium sulfate addition appeared to be the best one for biomass production comparable to those obtained using sodium nitrate as the conventional nitrogen source. Additionally, due to high cell growth observed in the cultivations, the demand for nitrogen was extremely high, reaching values around 12 mM per day. Taking into account that the inhibitory levels of ammonia is around 6 mM [13], in this case, considering the whole period of cultivation, the addition of 12 mM of ammonium sulfate per day in a single daily addition would probably lead to cell death. Thus, the daily addition was divided into eight times, which allowed a maximum cell concentration of approximately 14 g L-1 . In this work, a parabolic protocol for ammonium sulfate addition, in which the cells with 7% nitrogen was considered, led to biomass protein contents (35.6 ± 1.7%), comparable to that obtained in standard runs (35.5 ± 0.9%), thus demonstrating that the use of ammo­nium salts does not modify the cell composition.

Feeding time is a variable of great importance in the fed-batch process, where it is responsible for nutrient availability for the microorganisms. Concerning urea and ammonium salts for A. platensis cultivation, it is also very important to avoid any toxic effect of ammonia. A strict control of the feeding time in fed-batch culture in open ponds, at the same time, prevented the toxic effect exerted by excess ammonia or its loss by off-gassing [13]. As ammonia is the predominant nitrogenous species in the medium when ammonium chloride is used as a nitrogen source, an appropri­ate feeding time should be selected to maintain optimum ammonia levels through­out the whole cultivation. In fact, longer times limited growth because of the shortage of the nitrogen source, while shorter times affected the growth due to the occurrence of a displacement between nitrogen source supply and utilization [7] .

Bezerra et al. [7] reported that a short feeding time results in the partial loss of ammonia when using NH4Cl as nitrogen source and, consequently, growth limita­tion can occur. As evidenced by the increase in maximum cell concentration (Xm) from 1,111 to 1,633 mg L-1, a longer feeding time favored A. platensis growth. In addition, the run with the longest time to achieve the stationary phase exhibited a lower value of Xm (1,561 mg L-1), likely due to growth limitation by nitrogen begin­ning with the feeding step. This is in agreement with Carvalho et al. [13], who reported the existence of an optimum feeding time for cell production, below and above which biomass growth was affected by nitrogen source accumulation and consequent loss in the form of ammonia (feeding time of 12 days) or limitation (feeding time of 20 days) in the medium. Both situations led to decreases in Xm and suggested some discrepancy between biological demand and availability of the nitrogen source.

The fed-batch cultivation can be carried out as a repeated fed-batch process, in which once the cultivation reaches a certain stage after which it is not effective any­more, a portion of the culture medium is removed from the bioreactor and replaced by fresh nutrient medium. It allows keeping part of the medium in the reactor at the end of cultivation, reusing the exponentially growing cells for subsequent runs, cheaply ensuring high starting cell levels, and avoiding long stopping of the process. Moreover, it is expected to increase cell productivity, ensuring high cell growth rate. These features could be usefully exploited to evaluate the possibility of using this process with urea as a nitrogen source in large-scale cultivations. This process is characterized by removing a constant fraction of volume of culture at fixed time intervals and can be maintained indefinitely, where the volume is replenished to its maximum value by adding medium culture with appropriate flow rate [76, 115]. This type of process was employed by Matsudo et al. [62] to evaluate if urea could be successfully employed when using repeated fed-batch cultivation of A. platensis in open ponds. This study showed that the repeated fed-batch process using urea as a nitrogen source was suited for long-term A. platensis cultivation, during which the maximum cell concentration, the nitrogen-to-cell conversion factor and the kinetic parameters remained stable when using an appropriate ratio of renewed to total volume and experimental protocols of urea addition. Moreover, it should be noted that the biomass protein content was not influenced by the experimental conditions. The maintenance of maximum cell concentration during three consecutive cycles and the absence of contamination probably take place due to the well-known ability of this microorganism to grow well in saline and alkaline inorganic environments.

In conclusion, the fed-batch process is a useful tool for supplying carbon and nitrogen in cultivations of A. platensis under different photobioreactor configurations. For each specific carbon and nitrogen source added, it is necessary to evaluate the experimental conditions that lead to both desired cell growth and composition. Besides classical factors that affect photosynthetic cell growth, such as carbon source, nitrogen source, other nutrients, pH, temperature, light intensity and salin­ity, the typical parameters of the fed-batch process, such as feeding time, addition protocol and flow rate, should be evaluated. The comments presented in this chapter demonstrate that the use of inexpensive nitrogen sources, such as urea, ammonium salts and nitrogen-rich wastewaters can be used for A. platensis cultivation, with results that can be comparable to those with classical nitrate sources. The results also show that closed photobioreactor is useful for preventing ammonia loss during A. platensis cultivation. The best form of supplying organic carbon needs to be evaluated, where different strategies are necessary for each organic carbon source employed. In cases of using organic carbon, the process needs to be carried out under aseptic conditions. Considering inorganic carbon sources, CO2 has an impor­tant role in the growth of such cyanobacteria, where it needs to be added to the medium to maintain the carbon source level as well as pH. Finally, the fed-batch process was demonstrated to be useful for the production of A. platensis using CO2 from industrial plants, particularly from industrial alcoholic fermentations.

The antiviral family is one of the widest families of bioactive compounds isolated from marine sources, or at least one of the most studied. In this group, there are compounds like polysaccharides, terpenoids, proteins, sulfated flavones, and fatty acids. When measuring the antiviral activity, the general trend is to treat well-known mammal cells with the extract and then monitor the viral infection with the micro­scope. Huheihel et al. [63] used green monkey kidney cells (vero cells) treated with polysaccharides extracted from Porphyridium sp., the cell culture was treated with herpes simplex viruses. Each day, the cultures were examined for evidence of the cytopathic effect, defined as areas of complete destruction of cells or of morpho­logically modified cells and expressed as the percentage of damaged cells in the inspected fi elds.

But similar test can be done in vivo, Huheihel et al. [63] applying locally (eyes and mouth) Porphyridium extracts in rabbits and rats; later, the animals were exposed to the virus. Inflammatory effects, illness, and weight changes were recorded over a period of 4 weeks posttreatment.

Production of biogas from seaweeds and microalgae by AD was a subject of research starting in the mid-sixties [109, 110]. The first detailed large-scale study of seaweed cultivation and AD was performed in the Institute of Gas Technology by Chynoweth et al. [79, 111]. Substrate chemical composition and conversion parameters deter­mine the amount and composition of biogas generated in the ADP. The theoretical yield of biogas can be estimated by the Bushwell equation [112].

CcHhOoNn + yH2O ^ xCH4 + nNH3 + (c — x)CO2

where:

x = (4c + h — 2o — 3n — 2s) / 8
y = (4c — h — 2o + 3n + 3s) / 4

The theoretical methane yield from different algae is presented in Table 15.

Lipids have the lowest oxidation state and largest theoretical methane yield, which is more than twice the methane yield from proteins, glycerol, and carbohydrates. The theoretical methane yield correlates with the average carbon oxidation state of the substrate (Fig. 2) . Macroalgae with high carbohydrate content and cyanobacteria with high protein content are theoretically poorer feedstock for methane produc­tion while microalgae with high lipid content have higher potential methane yield.

Fig. 2 (a) Theoretical methane yield in relation to the average carbon oxidation state. circles— pure compounds; crosses—microalgae; pluses—macroalgae. (b) Mean theoretical methane yield and (c) C/N ratio with maximum and minimum values (base on Table 15)

Based on this observation, increasing the lipid content in algae is a promising approach for enhancing the methane yield. Another observation is that all microalgae have C/N ratio lower than optimal range for AD and high potential level for ammonia release. In contrast, several macroalgae and oil rich microalgae have a C/N ratio that is too high and possibly require addition of nitrogen for optimal AD conditions.

The application of molecular tools to microbial community structure analysis pro­vides insight about the development of a mature microbial community, its response to changes in substrate loading rate, and the influence of environmental factors [414]. This knowledge allows us to improve the design and control of the ADP.

Metabolic and Genetic Engineering of Anaerobic Microorganisms

Classical strain improvement is an evolutionary engineering technique that has been used for decades for the production of penicillin, amino acids, and many other industrial products, but it is time consuming and has limited applications [415, 416]. Attractive opportunities exist with metabolic and genetic engineering techniques to enhance biofuel production. Studies have been conducted with several organisms including Escherichia coli [417-420], Zymomonas mobilis [421-423], Klebsiella oxytoca [424,425], and S. cerevisiae [426-429].

Cellulolytic Clostridium is one of the prospective organisms for metabolic engi­neering in an anaerobic digester. Hydrolysis of polymeric organic compounds is often the limiting step during biodegradation of algal biomass. Hydrolysis of algal cell walls is difficult due to its heterogeneous structure composed of carbohydrates, glycoproteins, and lipids. Guedon and colleagues applied metabolic engineering to produce a recombinant Clostridium cellulolyticum that exhibited 150% higher cel­lulose consumption and 180% larger cell dry weight in comparison to the wild strain [430]. Another possible strategy is the expression in Clostridia of the algal cell wall degrading enzymes from wild strains that naturally use algal biomass as a substrate. Saccharophagus degradans is able to degrade at least ten distinct com­plex polysaccharides from diverse algal, plant, and invertebrate sources and can be an outstanding source of degrading enzymes [431, 432].

From the previous discussion, it is obvious that hydrate deposits that are being con­sidered as production targets must have the following attributes:

• Confirmed presence of high hydrate saturation SH.

• Occurrence within sediments of sufficient reservoir quality.

• Site accessibility through proximity to existing infrastructure.

• Access to gas markets through pipeline availability.

Here we discuss the features and attributes of hydrate deposits that are likely targets for gas production, and we analyze the geologic and engineering factors that control their ultimate resource potential.