Biodiesel is generally composed of several fatty acid esters including C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20 and C22:1. After oil extraction and purification, the exploitable fatty acid chains from the lipid undergo transesterification to produce fatty acid methyl esters (FAME). The transesterification reaction involves the use of an acidic or alkali catalyst mixed with methanol [36]. The methanol group attaches itself to the fatty acid chains via the bond cleaving activity of the catalyst to produce FAME and glycerol. The methyl ester (biodiesel) produced from this reaction after glycerol separation is crude, hence must be washed, dried and decon­taminated so that all water and particulates within the biodiesel are removed. The purified biodiesel must comply with the regulatory standards set by the Fuel Quality Standards Act 2000.

Microalgae are a class of microorganisms that number in thousands of species, which are present in a wide variety of ecosystems and live in a large variety of envi­ronment and environmental conditions. Under particular conditions, certain species produce large amounts of lipids and free fatty acids, compounds that are the basic raw materials for the production of biodiesel.

Other biofuels, such as bioethanol or higher alcohols, can also be obtained through the hydrolysis and fermentation of the algal biomass.

Alternatively, microalgal biomass can be gasified or pyrolysed to produce a range of other biofuels (biomass-to-liquid), hydrocarbons, biogas, and even biohy­drogen. Another possibility is the production of high value chemicals, such as pig­ments, proteins, and nutraceuticals, simultaneously with the production of biofuels, thus diversifying the sources of income and improving the overall profitability.

In addition to the aforementioned advantages, the cultivation of microalgae opens new possibilities for its integration within existing or future processes, such as wastewater and flue gas treatments, removal and sequestration of GHG, in particular
carbon dioxide, with the potential for reducing additional environmental impacts. In fact, various studies demonstrated the potential use of microalgae for pollution control, and production of useful products, such as commodity chemicals, and energy cogeneration combustion [5, 12]. Thus microalgae can be environmentally sustainable, cost-effective and profitable for the production of biofuels and other bio-products. In a broader sense, microalgae can be seen as an important part of a biotechnology supply chain that can produce many of the basic chemicals necessary for the development.

In this study the system boundary (Fig. 3) is defined to include the supply chain stages of microalgae biodiesel [11], from algal cultivation and further processing to biodiesel production, assuming that no additional products are obtained from the process. Please note that a consumption step is absent as it is the same as for other biofuels. Therefore, the main differences will occur in the first stages of each fuel supply chain.

Although microalgae have similar oil content to other seed plants, there are significant variations in the overall biomass productivity, the resulting oil yield, and biodiesel productivity, with a clear advantage for microalgae. Also, the growth and harvest of microalgae needs much less land area than other feedstocks of agricul­tural origin, up to 49 or 132 times less when compared to rapeseed or soybean crops, respectively, for a 30% (w/w) oil content in algae biomass [11]. Also, they can reproduce themselves using photosynthesis to convert solar energy into chemical energy, completing an entire growing cycle every few days.

As different algae species have similar efficiencies concerning fuels production, the selection of the most adequate species should take into account their character­istics and other factors, such as the quantity of nutrients available, solar irradiation, as well as other environmental conditions t 11t • From a practical point of view, microalgae are easy to cultivate, can grow with little or no attention, often using water unsuitable for human consumption, and it is easy to obtain the necessary nutrients [15]. They need not only nutrients (nitrogen and phosphorous), which are vital for the growth of algal biomass, but also adequate operating conditions (oxy­gen, carbon dioxide, pH, temperature, and light intensity). By manipulating these operating parameters one can easily control algal biomass growth and the composi­tion of the algal populations even at a larger scale. De Pauw et al. [2] state that

Fig. 3 System boundary including the supply chain stages of microalgae biodiesel

experience has repeatedly shown that properly managed algal cultures are quite resistant and that infections are often an indication of poor culture conditions.

Combined with their ability to grow under harsher conditions, and their reduced needs for nutrients, microalgae can be cultivated in areas unsuitable for agricultural purposes (on marginal or nonarable land), independently of the seasonal weather changes. This greatly reduces the competition for arable soil with other crops, in particular for human consumption, and can open up new economic opportunities for arid, drought, or salinity-affected regions [18 ] . Moreover, they are becoming an alternative oil source with favorable oil yields and much higher growth rates when compared to conventional biodiesel feedstocks.

Currently, much research effort is being focused on the algal production units, as in most cases it represents the key step that ultimately determines the economic viability of the process [11] . Microalgae cultivation units can be open or closed production systems, operated in multiple batch or continuous modes. These varia­tions depend on the microalgae species selected, the expected environmental condi­tions, nutrients available, and the possibility to combine the microalgae growth with a pollution control strategy of other industry, for example, for the removal of CO2 from flue gas emissions or the removal of nitrogen and phosphorus from a wastewa­ter effluent. If closed cultivation systems with minimal evaporation are used, con­siderable savings in water consumption can also be achieved.

Some limitations on second generation biofuels from microalgal culture systems have been identified in the harvesting process (representing 20-30% of the total production cost) and in the supply of CO. for a high efficiency production ]4]. A suitable harvesting method must be able to process large volumes of algal biomass and may involve one or more solid-liquid separation steps, such as sedimentation, centrifugation or filtration. Sometimes, an additional flocculation step is required, due to the need to remove large quantities of water. Drying and cell disruption for release of the metabolites of interest also represents a major economic limitation to the production of low cost commodities (foods, feeds, fuels) and also of higher value products (b-carotene, polysaccharides). Most common drying methods include spray-drying, drum-drying, freeze-drying and sun-drying, but the last one is not very effective because of the high water content of algal biomass. After drying, solvent extraction of lipids can be done directly from the lyophilized biomass using hexane or ethanol [11].

Even though, biodiesel feedstock’s can vary significantly, the biodiesel produc­tion process currently used at industrial scale is the alkali-catalyzed transesterification process where triglycerides and an alcohol (usually methanol) react in the presence of an alkali catalyst (e. g. NaOH or KOH) to obtain fatty acid methyl esters (biodie­sel) [11]. Also, depending on the oil acidity one or two process steps have to be performed, where in a first step, the level of free fatty acids is reduced to below 3% by acid-catalyzed esterification, using methanol as reagent and sulphuric acid as catalyst, for a portion of the methyl ester biodiesel and, in a second step, triglycer­ides in product from the first step are transesterified with methanol by using an alkaline catalyst to produce more of the same biodiesel and glycerol.

Once the microalgal culture is harvested from its cultivation system, it exists as a dilute aqueous suspension (between 0.1 and 1 wt.%) and needs to be concentrated for downstream processing. Solid-liquid separation techniques (centrifugation, filtration, flocculation) are typically used to dewater the culture up to a solid con­centration of 10 wt.% [9].

The dewatered microalgal paste then undergoes a pre-treatment step intended to enhance the efficiency of subsequent chlorophyll extraction. The pre-treatment can be performed in multiple steps or as a single process. As one alternative, the semi­wet paste is completely dried and the resulting biomass is milled into powder of uniform size. Residual water in the paste needs to be removed as it is known to act as a barrier which impedes chlorophyll transfer from the microalgal cells into the extracting solvent. As another alternative, the paste can be exposed to disruption meth­ods which destroy the microalgal cellular structures and force the release of intracel­lular chlorophylls to the surrounding medium. Exposing microalgal cells to mechanical cellular disruption methods (grinding, homogenization, and sonication) prior to sol­vent extraction has been found to increase final chlorophyll yield [28, 44, 49]. Simon and Helliwell [49] found that, without preliminary disruption, only a quarter of the available intracellular chlorophyll a can be extracted from microalgal cells.

As marine algae can carry on photosynthesis, they are able to synthesize all vitamins that high plants produce. The vitamin profile of algae can vary according to algal species, season, alga growth stage, and environmental parameters. The edible algae (especially of Porphyra spp.) contain large amounts of water-soluble vitamin C and B complex, and the fat-soluble vitamin A and E [71] . Algae are a good source of pro-vitamin A (see Sect. 4.1).

Algae provide a worthwhile source of vitamin C. The levels of vitamin C average 500-3,000 mg/kg of dry matter for the green and brown algae, which are compara­ble to concentration in parsley, blackcurrant, and peppers; whereas the red algae contain vitamin C levels of around 100-800 mg/kg [16]. Vitamin C is of interest for many reasons: it strengthens the immune defense system, activates the intestinal absorption of iron, controls the formation of conjunctive tissue and the protidic matrix of bony tissue, and also acts in trapping free radicals and regenerating vita­min E [16].

Brown algae contain higher levels of vitamin E (23-412 mg/kg of dry matter) than green and red algae (8 mg/kg of dry matter). H. elongata presents high levels of a-tocopherol as demonstrated by Sanchez-Machado et al. [160]; for example, the content of a-tocopherol in H. elongata dehydrated (33 mg/g dry weight) was con­siderably higher than H. elongata canned (12.0 mg/g dry weight), which clearly indicates the important effect of the processing on this compound. The highest lev­els of vitamin E in brown algae are observed in Fucaceae (e. g., Ascophyllum and Fucus sp.), which contain between 200 and 600 mg of tocopherols/kg of dry matter [96]. The red microalga Porphyridium cruentum also presents high levels of tocoph — erols as demonstrated by Durmaz et al. [30]; for example, the contents of a — and g-tocopherols were 55.2 and 51.3 mg/g dry weight, respectively. These tocopherols (vitamin E) are lipid-soluble antioxidants that are considered essential nutrients because of their ability to protect membrane lipids from oxidative damage [193]. Vitamin E has effect in the prevention of many diseases, such as atherosclerosis, heart disease, and also neurodegenerative diseases, such as multiple sclerosis [68, 73], thus also making it a very interesting functional compound. Generally, brown algae contain a-, b-, and g-tocopherols, while green and red algae contain only a-tocopherol [16]. Mendiola et al. studied the possible use of SFE to obtain fractions enriched with vitamin E from S. platensis [110].

Algae are also an important source of B vitamins; for instance, algae contain vitamin B1 2, which is particularly recommended in the treatment of the effects of aging, of chronic fatigue syndrome and anemia. Algae are also one of the few veg­etables sources of vitamin B12. U. lactuca can provide this vitamin, in excess of the recommended dietary allowances for Ireland fixed at 1.4 mg/day, with 5 mg in 8 g of dry foodstuff [36] . Spirulina is the richest source of B12 and the daily ingestion of 1 g of Spirulina would be enough to meet its daily requirement [196]. This may provide an alternate source of vitamin B12 for vegetarians or vegans.

On a dry matter basis, thiamine (vitamin B1) content ranges from 0.14 mg/g in dried H. elongata to 2.02 mg/g in dried Porphyra. and riboflavin (vitamin B.) content varies between 0.31 mg/g in canned H. elongata to 6.15 mg/g in dried Porphyra [163]. The amount of folate (as folic acid or vitamin B9) in the algae stud­ied by Rodrfguez-Bernaldo de Quiros et al. [153] (H. elongata, Laminaria ochro — leuca, Palmaria spp., U. pinnatifida, and Porphyra spp.) ranged from 61.4 to

161.6 mg/100 g of dry matter.

In conclusion, the algae have an original vitamin profile, which might comple­ment the vitamin profiles of land vegetables.

Algal productivity is limited by several factors that can be classified as physical, biochemical, ecological, and operational (Table 21) [53]. Cultivation and harvesting technologies are very dissimilar for macro — and microalgae due to significant differences

in physical or biochemical characteristics. Macroalgae are usually cultivated in marine nearshore and offshore zones, cages, ponds, tanks, or spray systems [173­178]. Microalgae are grown generally either in freshwater or brackish open ponds, closed photobioreactors, fermenters, or hybrid systems [179-188].

Fertilizers and inorganic chemicals are the major costs associated with intensive algal production systems. As example, producing 1 kg of biodiesel in fresh water requires 3,726 kg water, 0.33 kg nitrogen, and 0.71 kg phosphate [449]. On the other hand, algae and cyanobacteria play a role in self-purification of water bodies and in wastewater treatment by direct assimilation of simple organic compounds [294,450] and nutrients [451-456], removal ofheavy metals [388,453,457-459], and finally providing oxygen for organic matter oxidation by heterotrophic bacteria. Wastewater stabilization ponds with naturally occurring algal flora are widely used in developing countries and local sewage systems [460, 461]. Closed coastal areas, such as bays, fjords, and lagoons near urban and agricultural runoffs. are potential systems for cultivation, harvesting, and utilization of macroalgae for biomethane production [177, 412]. Golueke and Oswald first suggested the combination of wastewater treatment with production of algae to yield a biofuel [109, 110, 462, 463]. Coupling the treatment of nutrient-rich wastewater with algal growth followed by conversion to methane represents a potentially attractive biofuel production pro­cess with reduced impact on the environment [464-468]. Moreover, mixotrophic microalgal growth is attractive due to induction of lipids accumulation in algal cells.

Of the three main dissociation methods, depressurization appears best suited to the conditions of Class 1 deposits because of its simplicity, technical, and economic effectiveness, and the fast response of hydrates to the rapidly propagating pressure wave [129, 125]. Hong and Pooladi-Darvish [68] applied constant-P depressuriza­tion at a well at the center of a GH reservoir, and analyzed the sensitivity of the continuously declining production to various properties and operational conditions. They reported that, at the end of the first year of production, about 48% of the pro­duced gas had been replenished by hydrate-originated CH4 , thus confirming the technical feasibility of production from hydrates using conventional technology.

Moridis et al. [129] conducted a long-term (10-30 years) study of constant-rate (=0.81944 ST m3/s = 2.5 MMSCFD) production from Class 1 hydrate deposits. To describe the contribution of gas released from hydrate to the production stream, they introduced the concepts of Rate Replenishment Ratio (RRR) and Volume Replenishment Ratio (VRR). RRR is defined as the fraction of the gas production rate at the well(s) that is replenished by CH4 released from hydrate dissociation. VRR is defined as the fraction of the cumulative gas volume produced at the well(s) that is replenished by hydrate-originating CH4. During the 30-year production period, the VRR increases continuously to a maximum of about 0.74, and the corresponding RRR is 0.54 (Fig. 11 — [129]). The desirability and the great production potential of such deposits are obvious. The evolution of the SH distribution over time is shown in Fig. 12. Production from these Class 1 deposits (a) involved conventional technolo­gies and (b) necessitated continuous heating of the wellbore to prevent hydrate for­mation and plugging [129]. Note that the use of horizontal wells does not confer any practical advantages to gas production from Class 1 deposits [137].

A fundamental barrier to the potential utilization of methane from gas hydrates to serve future energy needs relates to our current limited understanding of the GH potential response to production activities and the associated environmental impacts. These impacts will include many issues that typically face current oil and gas explo­ration and production activities; including ground subsidence related to production from shallow, unconsolidated reservoirs, land and air impacts from drilling and pro­duction activities, and disposal of produced waters. Ultimately, despite the fact that GHs may be a major new source of clean-burning gas, it will remain difficult to expect public acceptance of large-scale gas hydrate production in the absence of a larger understanding of the GH role in the natural environment. This is particularly the case given the recognition that naturally occurring GH could provide a poten­tially deleterious feedback to ongoing climate change.

Gas hydrate is known to be an enormous storehouse of organic carbon with pro­found potential linkages to global carbon cycling and global climate [3, 38]. GHs are not a significant source of atmospheric greenhouses gases at present [72], but there is evidence in the geologic history attesting to the impact of methane from gas hydrate on global climates (e. g., [39]). This impact is enhanced by the powerful “greenhouse effect” of CH4 than CO2 (about 20 times larger) despite a short residence in the atmo­sphere [72]. Recent reports suggest that ongoing CH4 releases from shallow marine gas hydrates (particularly at high latitudes) may be linked to warming climates [150, 156, 158,159,212], although there are limited data for confirmation.

At present, the targets of gas hydrate production research are those accumula­tions in the Arctic or beneath the ocean floor that are housed in deep sand reservoirs bounded by nearly impermeable boundaries. As such, they are beyond the reach of potential climate-related temperature changes and are not expected to pose an envi­ronmental hazard as sources of methane release into the atmosphere. These repre­sent perhaps a significant resource volume, but is likely a very small percentage of the total in-place volume of methane associated with gas hydrates [9]. Thus, it is a critical task to assess the real potential impacts associated with only this small subset of resources that occupy the peak of the total gas hydrate resource pyramid [10].

Desirable production targets involve deeper, warmer sandy GH deposits [136] . Such deposits are closer to the stability conditions (requiring less energy for disso­ciation) and are housed in sediments of increased mechanical strength bounded by low-permeability strata. These settings render the deposits amenable to production through standard borehole-based techniques, reduce the tendency for sand produc­tion, and are less likely to affect the integrity of the overlying sealing lithologies. Environmentally invasive approaches, such as mining of sea floor and shallow sub­sea-floor deposits are not a consideration. This is not only because of regulatory restrictions (as such operations would harm protected complex and poorly under­stood biological communities that hydrates are known to host) but also because of the economic hopelessness of such a venture (given the resource leanness of such deposits and the astronomical cost of such sea floor operations). In other words, not the targets of gas hydrate production are generally environmentally insensitive.

An issue that deserves attention is the ability of the lithologic seal overlying GH reservoirs to contain the dissociated gas, which is known to accumulate at the top of the formation [136, 131, 132] . For shallow and unconsolidated reservoirs, the possibility of failure of the top seal is a serious consideration. Therefore, all respon­sible production test scenarios currently envisioned include plans to actively monitor the movement of the dissociation front and of the released gas accumulation in the reservoir [217].

Gas hydrate exploration and production activities will be prone to many of the same potential environmental impacts as conventional oil and gas production. A key issue will be ground subsidence: in the marine setting, this may mean assessing the risk for seafloor failure on slopes or other issues that may compromise the integrity of sea-floor infrastructure. In the arctic, this relates to preserving the integrity of the permafrost. Careful selection of drill sites and management of production processes, such as sand control procedures, are expected to be adequate to address such issues. Another issue may be water disposal. Gas hydrate production may result in significant volumes of produced water. Although the dissociated hydrate water will be virtually fresh, there will be an inevitable mixing with formation waters that will result in the production of brackish, non-potable water, which is anoxic and poten­tially harmful to chemosynthetic communities if released near the ocean floor. Such water will need to be handled and disposed of properly. However, none of these issues are unique to gas hydrates—and methods are currently being employed in a variety of settings to deal with even more acute impact issues.

6 Summary

Gas hydrates are a vast resource with a global distribution in the permafrost and in the oceans. Even if a conservative estimate is considered and only a small fraction is recoverable, the sheer size of the resource is so large that it demands evaluation as a potential energy source. Although difficulties exist, the development of hydrates into an energy source appears to have acquired its own global dynamic, with increased levels of international awareness, several national and international pro­grams investigating the feasibility of the endeavor, and heightened levels of activity. There is a concerted international effort to determine the technical and economic feasibility of production from gas hydrates.

Production from gas hydrates faces significant challenges because of the hostile environments in which they exist. The difficulty of access, the significant cost of related work, and the need for success in the first attempt at producing gas from this unconventional source have led to the development of a rational approach to priori­tize potential targets. By need and design, the first attempts to produce gas from hydrates will be limited to the few relatively well-characterized sites with proven resources.

Numerical simulation plays a critical role in the effort to assess the production potential of hydrates. While the dearth of field data has not allowed the full valida­tion of numerical codes, the scientific consensus is that the models generally account for the important physics of the problem, and that validation and calibration (rather than adequacy of the numerical code capabilities) will be a constraining factor in the assessment of the hydrates as an energy resource. A review of the data needs for the implementation of the numerical models indicates that, while knowledge gaps exist, these are being addressed, or can be adequately addressed by sensible approxima­tions. Sensitivity analyses can overcome the scarcity of data needed by the simulators by bounding the potential solutions. Literature review provides strong indications that gas hydrates from a variety of types of deposits (even ones considered unpro­ductive a few years ago) can yield large amounts of gas at high rates over long periods using conventional technologies. This bodes well for the production poten­tial of this unconventional resource.

The challenges and uncertainties facing commercial gas production from hydrates touch upon technical, economic, and environmental issues, and include (1) the assessment of in-place vs. resource vs. producible fractions of the GH resource, (2) the development and evaluation of methodologies for identifying suitable pro­duction targets, (3) the sampling of HBSs, sample analysis, and interpretation of the results, (4) the analysis and interpretation of geophysical surveys of GH reservoirs, (5) well-testing methods and interpretation of the results, (6) geomechanical and reservoir/well stability concerns in the course of gas production, (7) well design, operation, and installation appropriate for the particularities of GH systems, (8) field operations of production, (9) extending production beyond sand-dominated GH reservoirs, (10) monitoring production and geomechanical stability, (11) labo­ratory investigations and practices in support of gas production analysis, (12) fun­damental knowledge of hydrate behavior, (12) the economics of commercial gas production from hydrates, and (13) the associated environmental concerns.

Acknowledgments The contributions of G. J. Moridis, T. Kneafsey, J. Rutqvist, M. Kowalsky, and M. Reagan were supported by the Assistant Secretary for Fossil Energy, Office of Natural Gas and Petroleum Technology, through the National Energy Technology Laboratory, under the U. S. Department of Energy, Contract No. DE-AC02-05CH11231.

We have chosen three potential biofuel molecules as targets for which we will meta — bolically engineer R. eutropha (Fig. 4). We have targeted three classes of biofuel molecules: butanol, which will be derived from modifications to the PHB pathway; farnesene, which will be produced by introduction of a heterologous pathway to produce isoprenoids; and long-chain alkenes, which will be produced by modifying the fatty acid biosynthesis pathway and introducing genes for alkene biosynthesis. We have genetically modified wild-type R. eutropha to knock out the genes associ­ated with the PHB biosynthesis pathway [15]. We are comparing biofuel production from plasmid-encoded heterologous pathways in the wild-type strain with produc­tion in the PHB- strains.

Pyrolysis produces a hydrocarbon fuel composed by a minor amount of polar pyrolysis products. For this reason, from an operative point of view, pyrolysis could be considered as an “extraction process” for hydrocarbon-like substances and thus comparable with traditional solvent extraction.

Comparing traditional organic solvent extraction (with n-hexane, chloroform, and methanol) and thermochemical process, pyrolysis allows obtaining 90% of hydrocarbon-like materials of feedstock, as random length Cn-C31 alkanes and alkenes. As shown in Fig. 17, pyrolysis “extracts” selectively hydrocarbon-like material and produces very apolar oil that could be probably used directly as fuel, thanks to a similar composition with diesel fuel. On the other hand, traditional solvents extract more algal oil than pyrolysis, but this oil is heavier, due to presence of high molecular weight ether lipids. Moreover, the oil extracted with traditional solvents is richer in polar lipids, detrimental for fuel quality and solubility in standard fuels.

One of the most important drawbacks of pyrolysis process is that biomass has to be dried (up to 50% of moisture) before pyrolysis, whereas, as shown above, solvent extraction can be applied directly on wet materials. Nevertheless, pyrolysis results in an interesting solvent-less technique if microalgae can be easily collected and pelletized, as in the case of colonial B. braunii culture.

2 Conclusions

Different methods for extracting biofuel from B. braunii were tested. Both SPS and solvent-less thermochemical extraction resulted effective for the purpose. This is mainly due to B. braunii intrinsic properties. In comparison with traditional organic solvents, SPS have the interesting feature of being effective also on aqueous samples, without dewatering biomass. On the other hand, pyrolysis, in spite of the need to operate with relatively dry samples, maximizes the output of the process due to the co-production of biochar, energy, and a higher quality fuel than that obtainable through solvent extraction.

Acknowledgements We acknowledge the Ministry MiUR and the University of Bologna (RFO program) for funding. We thank Prof. Rossella Pistocchi and co-workers for providing with

Botryococcus braunii samples.