By conducting the audit on the entire process, we wish to quantify the GHG captur­ing benefits achievable from this process. The quantified GHG emission saving can be used to calculate the economic benefits from the process and also its environ­mental impact.

Nitrogen is the second most abundant element, representing approximately 10% of the cell [30] . It is a key element for cell growth and reproduction, since it is an essential building block of nucleic acids and proteins.

Piorreck et al. [75] reported that higher nitrogen levels lead to an increase in biomass production. The high nitrogen concentration in cultures generates an upward trend in protein and chlorophyll levels in the cells. On the other hand, low nitrogen concentrations lead to a decrease in the level of these compounds, and also reduce the rate of cell division. There is also an increase in the percentage of total carbohydrates in the cells [89], and phycocyanin is degraded and used as a nitrogen source [11]. Less chlorophyll and carotenoids are produced under these conditions, providing color changes in the culture medium which tends to become yellow [26, 60, 89].

Numerous nitrogen-containing compounds can be used by different organisms as sources of nitrogen. These include, for instance, inorganic ions such as nitrate or ammonium salts and simple organic compounds such as urea, amino acids, and some nitrogen-containing bases. Additionally, roughly half of the cyanobacteria are in principle capable of fixing N2 [100], but in the genus Spirulina, only Spirulina labyrinthyformis is able to fix atmospheric nitrogen. It is a thermophilic species, isolated from hot springs, and has been reported to be capable of carrying out facul­tative anoxygenic photosynthesis at the expense of sulfides [23] .

Studies in the literature have shown that the use of nitrate as a nitrogen source ensures high A. platensis concentrations, which justifies the wide use of the stan­dard culture media of Zarrouk [118], Paoletti et al. [72] and Schlosser [92], which employ potassium nitrate or sodium nitrate.

When using nitrate as a nitrogen source, the microorganism needs to reduce it to nitrite, in a reaction catalyzed by the enzyme nitrate reductase, and then to ammonia, in a reaction catalyzed by the enzyme nitrite reductase [43] according to the needs of the cell. This reduction of nitrate to ammonia requires energy. In fact, comparison of the yield of growth on Gibbs energy obtained using either urea or KNO3 in batch and fed-batch processes, respectively, pointed to the preference of S. platensis for the former nitrogen source, likely owing to more favorable bioenergetic conditions [88]. Such behavior was very evident at the end of cultivation, likely because pho­tosynthesis efficiency decreased with increasing biomass concentration suggesting that biomass had used energy, under limited growth conditions, preferentially for maintenance rather than for growth [106], and consequently, it grows better with a nitrogen source that demands less energy. These facts point out that the fed-batch process carried out under optimal conditions does not hamper cell growth.

No negative effect of nitrate concentration during A. platensis cultivation has been reported. S. platensis cultivation using a fed-batch process with the addition of exponentially increasing mass flow of KNO3 as the nitrogen source was studied by Rangel-Yagui et al. [80]. These authors reported that cell growth was similar to that obtained with the batch cultivations using nitrate as the nitrogen source. Although lower cell concentrations were not observed for the cultivations with fed-batch addi­tion of KNO3, there is no need for the use of this process as opposed to batch cultiva­tion, since a batch process is easier to operate compared to a fed-batch process. It is worth mentioning that even nitrate nitrogen sources can be necessary to be added during the cell growth in cultivations with high final cell concentrations [41].

Alternative sources of nitrogen such as urea and ammonium salts can also be used to reduce production cost, replacing sodium or potassium nitrate, since urea and ammonium salts such as ammonium sulfate are widely used in agriculture.

Ammonia in alkaline medium is easily assimilated by the microorganism, since ammonia uptake involves simple diffusion followed by trapping through protona­tion, thanks to the membrane being permeable to ammonia but impermeable to ammonium ions [10] .

As noted before, urea is hydrolyzed to ammonia in alkaline culture medium [33] and/or by urease action [93]. Since ammonia can be toxic to microalgae and cyanobacteria at high concentrations, the addition of urea in a fed-batch process makes this nitrogen source a promising substitute for nitrate salts in photosynthetic microorganism cultivations.

If ammonium salts and/or urea are added in large quantities at the beginning of cultivation, there is excessive formation of ammonia, leading to cell death. In fact, growth inhibition or even cell death can take place when using ammonium at rela­tively high concentrations because of the toxic effect of ammonia [1, 6]. Moreover, batch cultivations of A. platensis in 500-mL Erlenmeyer flasks using ammonium chloride as a nitrogen source resulted in nitrogen limitation below 1.6 mM nitrogen, whereas above 6.4 mM there was excess ammonia, inhibiting cell growth [13]. The use of low starting levels of ammonium chloride for batch Spirulina sp. production proved to hinder growth because of nitrogen limitation, with consequent decrease in biomass yield compared to the use of nitrate [30, 91]. Confirming these findings, Faintuch [38], studying different nitrogen sources in Spirulina maxima cultivation with a batch process, found that the use of alternative nitrogen sources instead of nitrate, such as ammonium chloride and urea, was limited to low concentrations of nutrient, resulting in low cell concentration.

Particularly in the use of ammoniacal nitrogen sources for A. platensis cultiva­tion, the control of the nutrient flow can prevent inhibitory concentrations of ammo­nia in the culture medium [7, 13, 97]. The fed-batch addition of urea was shown to be an excellent protocol to ensure the optimum level of this compound in the growth medium, thereby allowing satisfactory S. platensis cultivation [33, 88, 106] and ammonium salts [7, 13, 41] . Despite the above inhibitory effect of ammonia, the fed-batch addition of ammonium salts or urea did in fact prevent any inhibitory effect, allowing such a cyanobacterium to reach a concentration comparable to that obtained with nitrate [13, 33, 41, 80].

A. platensis can grow with either nitrate or ammonium salts as the sole nitrogen source, but the simultaneous use of both is advantageous. The use of ammonium chloride alone as the nitrogen source in A. platensis cultures can lead to slightly lower cell concentration and biomass with reduced final contents of chlorophyll, lipids and proteins. On the other hand, the combination of potassium nitrate and ammonium chloride provides a way to deal with these issues. The presence of nitrate likely prevents a deficiency of nitrogen in cultures with ammonia, and the presence of ammonia reduces the amount of nitrate to be added, helping to reduce production costs. Moreover, the residual nitrate level in cultures with both nitrogen sources was much lower than that detected with one source alone, hence reducing the salinity, the environmental impact, and the disposal and/or the reutilization of effluent [84]. It would also be characterized as a fed-batch process with the use of two nitrogen sources, since one of them was fed during the microbial cultivation. These authors cultivated A. platensis in mini-tanks at 13 klux (156 mmol photons m-2 s-1) using a mixture of potassium nitrate and ammonium chloride as nitrogen source. The addi­tion of the total amount of potassium nitrate was made entirely at the beginning of the runs and ammonium chloride was added by daily pulse-feeding, using an expo­nentially increasing regime in order to avoid any inhibitory effect by ammonia or nitrogen deficiency. Higher maximum cell concentration was obtained in the culti­vation with both nitrogen sources supplied in comparison to that obtained with cul­tivations carried out with ammonium chloride or potassium nitrate, separately. Ammonia lost by volatilization during the cultivation [13] leads to nitrate acting as a reserve source of nitrogen when ammonia concentration in the culture medium is not sufficient, thus explaining the better results obtained with the two nitrogen sources combined.

Fed-batch process has also been employed to complement natural media. Costa et al. [31] supplemented the Mangueira Lagoon water with 1.125 mg L-1 of urea when S. platensis had reached the stationary phase (approximately 312 h), and this operation resulted in a 2.67-fold increase in the final biomass concentration.

Wastewater can also serve as nitrogen source. Olgufn et al. [70] evaluated the annual productivity of Spirulina (Arthrospira) and its ability to remove nutrients in outdoor raceways treating anaerobic effluents from pig farm wastewater, diluted in a mixture of sea and fresh water, under tropical conditions. The average productiv­ity of semi-continuous cultures during summer was 15.1 g m-2 d-1 with a pond depth of 0.20 m. Under the conditions studied, NH4-N removal was in the range of 84-96% and P removal in the range of 72-87%, depending on the depth of the culture and the season. These findings show that the Spirulina can be used for ammonia removal from wastewater in processes in which the wastewater is fed periodically.

Aiming to remove nitrogen and phosphorus in shrimp cultivation, Chuntapa et al. [22] evaluated the simultaneous cultivation of S. platensis and this crustacean. In the absence of S. platensis, ammonium and nitrite concentrations ranged from 0.5 to 0.6 mg L-1 , while nitrate concentrations ranged from 16 to 18 mg L-1 by day 44. Considerable variability in nitrogen concentrations occurred when the S. platensis was not harvested from the ponds. Semi-continuous harvest of S. platensis reduced nitrate to 4 mg L-1, while ammonium and nitrite ranged from 0.0 to 0.15 mg L-1, respectively. Kamilya et al. [51] observed that the same microorganism could remove up to 92 and 48% of nitrogen and phosphorus, respectively, thus contribut­ing to the reduction of eutrophication of fish farm effluent. Olgufn [71] described a process using sea water together with pig farm wastewater for low-cost S. platensis production and an animal wastewater treatment as well.

3.2.1 Antihelmintic, Antifungal, and Antibacterial Activity

In terms of antibacterial and antifungal activity, several compounds have been described in extracts from algal origin. Compounds like phenols, indoles, pep­tides, steroidal glycosides, terpenes, fatty acid, and so on. Basically, the method consists on letting the organism grow in the presence of the extract or compound. For example, Mendiola et al. [ 109] used a broth microdilution method to test the minimum inhibitory concentration (MIC) of Spirulina extracts on the growing of several bacteria and fungi. Tests were done in microwell plates, prepared by dis­pensing into each well culture broth plus inocula and 30 qL of the different extract dilutions. After incubation, the MIC of each extract was determined by visual inspection of the well bottoms, since bacterial growth was indicated by the pres­ence of a white “pellet” on the well bottom. The lowest concentration of the extract that inhibited growth of the microorganism, as detected as lack of the white “pellet,” was designated the MIC. The minimum bactericidal and fungicidal concentration was determined by making subcultures from the clear wells which did not show any growth.

Among antihelmintic compounds derived from algae, sesquiterpenes, like b-bisabolene, are the most actives. The most common method to measure its activ­ity is to grow the helminths (worms, i. e., Nocardia brasiliensis) in the presence of the alga extract. For example, Davyt et al. [22] used tissue-culture 24-well plates. They prepared dilutions in DMSO for each compound, in order to obtain the desired concentration after the addition of 10 qL into each well. The percentage of dead worms was determined on day 5 and corrected by controls and compared with synthetic drugs.

3.2.2 Anticoagulant Activity

Polysaccharides, especially sulfated polysaccharides, are the main anticoagulant compounds isolated from algae and microalgae. Its activity is commonly measured providing the compound in vivo and measuring in vitro how coagulant factors are varied. For example, Drozd et al. [27] administered fucoidans (5 or 10 mg/kg) into the jugular vein of male Wistar rats, collected the blood and measure the inhibition of Xa factor (anti-Xa — or aHa-activity) and thrombin (anti-IIa or aIIa-activity). Specific activity was calculated in U/mg by comparison of optical density of the test and standard solutions during hydrolysis of chromogenic substrates.

The primary treatment of biogas includes cooling, drying, and almost always remov­ing of hydrogen sulfide. More advanced applications of biogas require upgrading it to biomethane or removing carbon dioxide. The following methods are used for the removal of carbon dioxide from biogas: pressure swing absorption on zeolites, selective membrane separation, cryogenic separation, and biological or chemical fixation [104-106]. The typical technologies for biogas cleaning include scrubbing by solvents or an aqueous alkaline solution, absorption, and oxidation on solid sor­bents, chelation, precipitation in the form of poorly soluble metal sulfides, and bio­logical removal [105, 107, 108].

1.1.4 Biogas Utilization

The possible applications of biogas include:

• Heat or steam production via burning

• Electricity generation combined with heat and power production

• Usage as cooking gas instead of natural gas

• Usage as fuel for vehicles (upgrading to biomethane is necessary)

• Generation of electricity via fuel cells

• Production of chemicals

The C/N ratios for microalgae are in the range of 4-6. The addition of carbon rich cellulosic materials can balance the high nitrogen content. For example, addition of 25 and 50% of waste paper to a mixture of Scenedesmus spp. and Chlorella spp. resulted in a 1.59- and 2.05-fold increase in the methane yield (Fig. 17a) [410]. The optimal ratio between algal biomass (Scenedesmus spp. and Chlorella spp.) and waste paper was found to be 40% algae and 60% paper with corresponding C:N ratio equal to 22.6. The influence of the C/N ratio on the methane yield is shown in Fig. 17b. The authors also reported that paper addition stimulated cellulase activity in the anaerobic digester from 1.26 ± 0.14 mg/L-min (no paper added, C:N is equal to 6.7) to 3.02 ± 0.09 mg/L-min (50% paper, C:N is equal to 18).

Addition of A. maxima biomass to sewage sludge, peat extract, and spent sulfite liquor improved the VS reduction and methane yield (Fig. 18) [411]. Nutrient rich algal and cyanobacterium biomass can be added to nutrient limited waste products that cannot be digested as sole substrate.

Co-digestion of Macroalgae

The mix of Ulva and manure has a larger methane yield and production rate com­pared to pure Ulva biomass (Fig. 11c, d). Methane production from a mixture of alginate extraction residues and manure was lower compared to methane production from separate substrates [245]. Morand and coauthors speculated that co-digestion of different seaweeds can be problematic due to dissimilarity in digestion speeds [412]. But addition of Ulva to Sargassum tenerrimum (1-17 ratio) increased the methane yield and production rate [413].

As in the case of conventional hydrocarbon production, it is logical to expect that the first gas recovery from hydrate resources will occur where there is relatively easy site access and GH are concentrated [21]. Such sites and deposits constitute the first targets on which the attempt to produce gas from hydrates must be focused.

The analysis of Boswell and Collett [ 10] used relative prospects for future produc­tion as the criterion to identify several key tiers of GH resource categories within the context of a resource pyramid (Fig. 2). At the peak of the Gas Hydrates Resource

Arctic sandstones under existing infrastructure (-10 s of Tcf in place)

I— Arctic sandstones away from infrastructure (100s of Tcf in place)

Deep-water sandstones MOOOTcf in place)

Non-sandstone marine reservoirs with permeability (unknown)

Massive surficial and shallow nodular hydrate unknown)

Marine reservoirs with limited permeability

Fig. 2 Gas hydrates resource pyramid (left). To the right is an example gas resources pyramid for all non-gas-hydrate resources [10]

Pyramid (those resources that are closest to potential commercialization) are deposits that exist at high hydrate saturation SH within quality reservoir rock under existing Arctic infrastructure (e. g., in the Eileen trend of the Alaskan North Slope), estimated to represent 9.4 x 1011 m3 STP (=33 TCF) of gas-in-place. Modeling studies suggest that as much as 3.4 x 1011 m3 STP (=12 TCF) of that volume may be technically recov­erable. The second-from-the-top tier of hydrate resources is that of less well-defined accumulations that exist in similar geologic settings (discretely trapped, high-SH occurrences within high-quality sandstone reservoirs) on the North Slope, but away from existing infrastructure. The current USGS estimate for total North Slope resources is approximately 1.7 x 1013 m3 STP (=590 TCF) gas-in-place [22].

The next most challenging (third) tier of resources includes GH of moderate-to — high SH that occur within high-quality oceanic sandstone reservoirs. The most favor­able accumulations occur in the GOM in the vicinity of oil and gas production infrastructure [8]. Additional examples of this category of resource have been docu­mented from the Nankai Trough studies offshore Japan [47] and by the IODP Expedition 311 offshore Vancouver Island [162, 163]. All subsequent tiers are considered unat­tractive, and major technological advancements will be needed before production from such deposits is ever to become feasible [140, 125].

The results of examining and testing natural HBS cores in the laboratory environ­ment are dependent on the quality of the collected core, and the quality of the sub­sampling and analysis. Gas hydrate is stabilized by elevated constituent gas pressure, as affected by the temperature and inhibitor concentration in the pore water. Drilling often requires dense muds with high ionic strength, which can alter pore water chemistry and cause hydrate dissociation (e. g., [193]). Oil-based muds can be used to minimize pore water chemistry changes [12] . Pressure coring has been used to stabilize cores at their initial reservoir pressures [23, 28, 29] . and appropriate sampling equipment such as the instrumented pressure testing chamber [220-222] was developed. Field-based X-ray imaging and CT scanning, useful for understanding the core conditions and to identify representative locations for sam­pling and analysis, have been performed for pressurized and non-pressurized core [23,28,29, 67,81].

Preserving core for later examination requires careful handling. Many measure­ments require briefly removing a sample from hydrate-stable conditions to transfer it into a testing apparatus. Waite et al. [205] and Kneafsey et al. [84] reported that such handling results in hydrate redistribution that affects the mechanical, flow, thermal, and electrical properties of the sample. Freezing samples in liquid nitrogen preserves the hydrate and may be the best technique for maintaining the hydrate chemistry, but that can induce extensive fracturing (caused by large thermal stresses) and affect non-chemistry measurements [84]. X-ray computed tomography (CT) can be useful in the selection of undamaged and representative parts of a GH-bearing core for sampling. Prior to testing, a preserved core needs to be returned to its natu­ral conditions by imposing very mild thermal gradients and using materials that do not fail at the preservation temperatures (usually liquid N2).

Because of the GH sensitivity, it can be argued than an undisturbed sample has yet to be collected. Because of the importance of such samples in the evaluation of the feasibility of gas production, the subject needs further attention.

As archetypes of O2 tolerant hydrogenases, the R. eutropha enzymes have been extensively studied using various spectroscopic techniques and site directed muta­genesis (reviewed in [ 17, 36 ] ) . Although these studies have proven that direct manipulation of the hydrogenases is possible, it has also shown the difficulty of improving activity of the enzymes. Therefore, direct manipulation of the hydroge — nases to optimize the generation of energy and reducing equivalents for IBT produc­tion would be a significant challenge, and likely not a viable approach. In contrast, hydrogenase gene expression will be enhanced to assure that sufficient reducing equivalents will be available for optimal IBT yield.

To optimize IBT yield, it is important to balance the reducing equivalents gener­ated by the SH with the ATP synthesized by the MBH. Since carbon fixation will be maximized and the IBT synthesis pathway consumes two NAD(P)H molecules for every two molecules of pyruvate reduced to IBT, the hydrogenase activity balance will be shifted towards the SH.

3.2 Enhancement of Carbonic Anhydrase

Because of the inefficiency of the enzyme, the CO2 fixation by RuBisCO is likely the limiting step for an efficient production of IBT by R. eutropha. Enhancing expression and/or activity of CAs could help to increase the CO2 concentration in the cytosol, thereby limiting the competing oxygenation reaction by RuBisCO and increasing the CO2 flux through the CBB cycle and subsequently to IBT.

4 Outlook

Closed photobioreactors enable axenic cultivation of microalgae, maximal control of culture parameters, e. g., pH and temperature, and prevent water loss due to evap­oration, one major drawback of open pond cultivation systems [33].

Their solar light capturing surfaces consist of transparent materials with long shelf lives, e. g., poly(methyl methacrylate) (PMMA) [14, 41], borosilicate glass [28], or simply compartments of plastic film [31].

In general, three basic designs were developed on the basis of the aforemen­tioned considerations. These are bubble columns, flat plate, and tubular reactors (Fig. 6). Mainly implementation of a short light path length and the principle of light dilution determine the fundamental geometries of these reactors.

With regard to economic considerations, investment costs should not exceed 50 US$/m2 for biofuel production [30] and operating costs equally need to be mini­mized. The energy input is one important cost factor and likewise significantly influences the net energy gain. Table 2 gives an overview of power input of several reactors in outdoor experiments.

The main characteristics of three common reactor types are outlined in the fol­lowing sections. Furthermore, some variants, adapted to the extended demands, such as low cost and high productivity, are shown.

This section will explore the process engineering design for an industrial scale microalgal production plant for biodiesel production. The design will focus on four key process steps: biomass cultivation, dewatering, extraction and biodiesel produc­tion. Various unit operations for each process step are designed to compare their appropriateness, and recommendations are made to clearly define the unit opera­tions that best optimise the process.