Levulinic acid (4-oxopentanoic acid) is a high-boiling point, water-soluble biomass-derived acid that crystallizes at room temperature. Levulinic acid contains two reactive functional groups (single bondCdouble bond; length as m-dashO and single bondCOOH) that provides, as in the case of lactic acid, a rich chemistry to this compound [37]. Levulinic acid occupies a prominent place in the selected list of biomass platform molecules [11] since it is simply and inexpensively produced from lignocellulose wastes (paper mill sludge, urban waste paper, agricultural residues) by acid dehy­dration of C6 sugars [38]. Interestingly, equimolar amounts of formic acid

are co-produced along with levulinic during the dehydration process. As will be described below, this formic acid can be used as a renewable inter­nal source of hydrogen in the conversion of levulinic acid into advanced biofuels. Dehydration of sugars to levulinic acid is typically accompanied by unwanted polymerization reactions that produce intractable black in­soluble materials denoted as humins. These humins are typically burnt in the industrial process to generate heat and electricity. Interestingly, C5 sugars such as xylose (the main component of hemicellulose which typi­cally accounts for 20-30% of lignocellulose) can also serve as a source of levulinic acid. The process is not as straightforward as in the case of hexoses since it involves previous dehydration of xylose to furfural, sub­sequent hydrogenation to furfuryl alcohol and final hydrolysis of the latter to levulinic acid [39]. However, this route would benefit from an easier de­construction of amorphous hemicellulose compared to highly recalcitrant crystalline cellulose (the main component of lignocellulose and the source of C6 sugars) leading to potential operation at milder acidic conditions at which formation of humins is more controlled.

Among the different processes that have been developed for the large — scale continuous production of levulinic acid, the most promising tech­nology has been patented by the Biofine Corporation [40] and [41]. This approach utilizes a double-reactor system that minimizes the formation of by-products and the resulting separation problems. Lignocellulose is fed to the reactor along with a solution of sulfuric acid. The by-products of the process (furfural and formic acid) are condensed and separately col­lected, while solid humins are removed from the levulinic acid solution and collected as combustible wastes. This technology allows production of levulinic acid at low cost (0.06-0.18 €/kg) [42], making this compound suitable for use as a platform molecule. There are currently several plants operating with tons of waste cellulosic materials per day, both in the U. S. [42] and in Europe [43].

As shown in Fig. 3, levulinic acid possesses great potential for the pro­duction of advanced biofuels of diverse classes. Most of the routes involve intermediate formation of y-valerolactone (GVL), a stable and water-sol­uble biomass derivative with potential to be blended with gasoline as well as to serve as a precursor of polymers and fine chemicals [44] and [45]. The reduction of levulinic acid to GVL is thus a process with interest, and several routes involving different catalysts and hydrogen sources for this reduction have been explored in recent years. The most simple route in­volves utilization of carbon-supported noble metal catalysts under hydro­gen pressure [46], [47] and [48] which achieves near quantitative yields of GVL at mild temperatures (e. g. 150 °C) with slight deactivations with time on stream. The utilization of mild temperature conditions and non-acidic supports such as carbon is crucial to direct synthesis through 4-hydroxy — pentanoic acid which subsequently undergoes highly favorable internal esterification to the five member ring GVL compound [49]. The utilization of higher temperatures and/or acidic catalysts promotes dehydration of levulinic acid to a-angelica lactone. This compound polymerizes readily over acidic surfaces leading to loss of carbon as coke and severe catalyst deactivation.

As remarked above, the formic acid co-produced in the sugar dehydra­tion process can serve as a internal source of hydrogen for the reduction of levulinic acid to GVL [13], [50], [51], [52], [53], [54] and [55]. These technologies take advantage of the easy decomposition of formic acid into CO2 and H2 to generate an in situ source of this gas within the reaction system. Interestingly, the same materials used for reduction of levulinic acid to GVL are also able to catalyze decomposition of formic acid. In this sense, excellent GVL yields (96%) have been reported by Deng and co-workers in two different works involving homogenous [13] and het­erogeneous [51] Ru-based catalysts. This ability of Ru to quantitatively convert levulinic acid into GVL via formic acid decomposition has been utilized to expand this route to hexoses thereby allowing production of GVL from biomass sugars in a one-pot process and without hydrogen re­quirements [52]. The process, however, requires additional utilization of an acidic medium which serves as a catalyst for sugar dehydration, and GVL yields obtained, limited by the sugar dehydration process, are typi­cally modest (50%).

Since industrial manufacturing of levulinic acid involves treatment of biomass with aqueous sulfuric acid, it would be interesting to find a cata­lyst that can efficiently transform aqueous sulfuric acid streams of levu — linic acid and formic acid into GVL without the need for previous and waste-producing neutralization steps. In this sense, aqueous sulfuric acid solutions of levulinic and formic acids, obtained after acid hydrolysis of

solid cellulose, can be transformed into GVL with acceptable yields over Ru/C and Ru-Re/C catalysts [53] and [56]. Importantly, formation of GVL allows the design of strategies for the recycling of most of the sulfuric acid utilized for biomass depolymerization and sugar dehydration processes. Thus, alkylphenol solvents, with superior abilities to selectively extract GVL from aqueous sulfuric acid solutions, have been recently proposed for this task [57].

As summarized in Fig. 3, GVL presents high versatility to synthesize advanced transportation biofuels of diverse classes. The most direct route involves conversion into methyltetrahydrofuran (MTHF) via hydrogena­tion to 1,4-pentanediol over metal catalysts at moderate temperatures (250 °C) and subsequent dehydration of the diol to yield the cyclic ether [42]. The process takes advantage of the natural tendency of 1,4 diols to under­go dehydration/cyclation with temperature (AG = -73 kJ/mol for 1,4-pen — tanediol at 250 °C) to afford MTHF from levulinic acid with high yields (83%). MTHF is a hydrophobic molecule which, unlike ethanol, can be blended with gasoline up to 60% (v/v) without adverse effects on engine performances or gas mileage and can be distributed by existing pipeline for hydrocarbons without water contamination. MTHF is one of the com­ponents of the so-called P-series fuels which are approved by the US DOE for use in gasoline vehicles.

One route that is gaining interest in recent years involves transforma­tion of GVL into pentanoic acid. The process involves acid-catalyzed ring opening of GVL to pentenoic acid and subsequent hydrogenation of the latter over bifunctional (metal and acid) catalysts at moderate tempera­tures and hydrogen pressures [48] and [58]. The formation of pentanoic acid achieves reduction of the oxygen content of levulinic acid thereby producing a less-reactive intermediate which is more appropriate for new upgrading strategies to larger compounds. For example, Lange and co­workers [59] have used this route to produce the so-called valeric biofuels (i. e. alkyl valerates). Valeric biofuels can be used in conventional engines without any modification since they present similar energy-density, polar­ity and volatility-ignition properties than hydrocarbon fuels. The process is flexible in that by varying the alkyl chain length the fuels can be adapted to fit in both gasoline and diesel engines. The main drawback of this tech­nology lies in the need for external alcohol source for esterification.

Alternatively, liquid hydrocarbon fuels appropriate for gasoline and diesel applications can be produced via 5-nonanone, the ketonization product of pentanoic acid (Fig. 3). Interestingly, 5-nonanone can be pro­duced in high yields (70%) from aqueous GVL over a single bed of Pd/ Nb2O5 catalyst in which niobic support catalyzes GVL ring opening and pentanoic ketonization reactions [48]. Nonanone yield can be increased to almost 90% by using a double-bed reactor configuration with Pd/Nb2O5 + Ce05Zr05O2 operating at two different temperature zones (325 and 425 °C) which allows for optimum control of reactivity [53]. As shown in Fig. 4, the C9 ketone, which is obtained in high yields stored in an organic layer that spontaneous separates from water, can be upgraded to liquid hydrocarbon fuels by means of well-known petroleum-based chemistry. For example, by consecutive cycles of hydrogenation/dehydration over a bifunctional metal-acid catalyst such as Pt/NbPO4, 5-nonanone can be converted into n-nonane which possesses excellent cetane number, lubric­ity and cloud point properties to be used as a blender agent for winter die­sel applications. Alternatively, 5-nonanol, obtained by the hydrogenation of the C9-ketone, can be dehydrated and isomerized in a single step over an USY zeolite catalyst to produce a mixture of branched C9 alkenes with the appropriate molecular weight and structure for use in gasoline after hy­drogenation to the corresponding alkanes [53]. Larger hydrocarbons such as those required for diesel vehicles can be produced from the nonanone stream by means of oligomerization reactions of the previously formed C9-alkenes over an acid catalyst such as Amberlyst 70 [60]. This process allows conversion of approximately half of the mass of GVL into C18 alkenes which retain more than 90% of its energy content.

Aviation requires fuels with high energy density (to allow storage of large amounts of fuels in tanks with a size determined by aircraft design) and with extremely low cloud points (to ensure operational use at high altitude temperatures). Branched hydrocarbons in the C9-C16 range meet those requirements and, consequently, routes for the production of these compounds from biomass sources are highly valuable. Recently, a promis­ing route to upgrade aqueous solutions of GVL into jet fuels through the formation of C4 alkenes has been developed by Bond et al. [23] (Fig. 3). In this process, GVL undergoes decarboxylation at elevated pressures (e. g. 36 bar) over a inexpensive silica/alumina catalysts, producing a clean gas

stream composed of butenes isomers and CO2. This gaseous stream is then passed over an acidic catalyst (H-ZSM5, Amberlyst) that achieves oligo­merization of butenes yielding a distribution of branched alkenes centered at C12 suitable to be used as jet fuels after hydrogenation. This technology presents important economic and environmental advantages: (a) minimum amounts of external hydrogen are required in the process, (b) precious metal catalysts are not required, (c) a gas stream of pure CO2 is produced at the elevated pressures, thereby permitting effective utilization of se­questration or capture technologies to mitigate greenhouse gas emissions.

H2 is the typical reagent employed for levulinic acid deoxygenation to advanced biofuels, and efforts are currently aimed to obtain this gas from biomass sources. Electricity, based on renewable wind or solar pow­er, could be alternatively used for electrochemical deoxygenation of bio­mass to fuels. Nilges and co-workers have recently developed an approach for the electrochemical conversion of levulinic acid into octane [61]. As shown in Fig. 3, the process involves the combination of electroreduction of aqueous levulinic acid into pentanoic acid and subsequent oxidative Csingle bondC coupling of the latter (e. g. Kolbe reaction) to octane. Apart from the spontaneous separation of octane from the water medium, the use of electricity gives this process a number of green characteristics includ­ing the use of mild conditions (room temperature and water solutions), the replacement of any reducing chemical agent by immaterial electrons, and the minimization of waste generation. Although this preliminary work still has to solve some issues such as the effect of potential impurities in levu — linic acid (from biomass depolymerization steps) on the electrochemical reactions and the design of more efficient electrodes for large-scale ap­plications, it represents an interesting approach for converting the unused electricity generated during overproduction cycles (typical of fluctuating electricity production profiles of wind and photovoltaics) into a storable biofuel form.

Deoxygenation of levulinic acid to advanced biofuels can be carried out without utilization of hydrogen by means of a pure thermal pyrolysis treatment. The process is denoted as thermal deoxygenation (TDO) and involves previous formation of calcium levulinate and heating of the latter to temperatures ranging 350-450 °C under inert atmosphere [62] (Fig. 3). At these conditions, calcium levulinate simultaneously con­denses (by ketonization) and deoxygenates (by internal cyclation and dehydration) leading to the production of a broad product distribution of cyclics and aromatics with very low oxygen content. Production of aromatics, which are valuable components of gasoline and jet fuels, can be maximized by operating at higher temperatures. The process has been recently improved by addition of equimolar amounts of calcium formate which serves as an in situ hydrogen source allowing a deeper TDO pro­cess leading to the formation of a petroleum-like oil which could be further processed in existing refinery facilities [63].

7.4 CONCLUSIONS

Our society is highly dependent on fossil fuels, which are non-renewable and contribute to global warming. The conversion of biomass into fuels for the transportation sector can help to partially alleviate this reliance. Biodiesel and bioethanol, the main biofuels used today, present serious compatibility issues which can be overcome by the production of ad­vanced biofuels such as higher alcohols and green hydrocarbons which are fully compatible with our existing hydrocarbons-based transportation infrastructure.

However, working with complex biomass feedstocks is difficult and ap­proaches based on the formation of simpler and more stable intermediate derivatives, denoted as platform molecules, have been shown to be effec­tive for efficient biomass conversion to fuels and chemicals. Lactic acid and levulinic acid are two of these relevant biomass derivatives that can be trans­formed into advanced biofuels by a number of catalytic routes involving deoxygenation reactions combined with Csingle bondC coupling processes. The present paper offers a state of the art overview of the most relevant cata­lytic strategies available today for this paradigmatic conversion.

Sedimentation of algal biomass is a further method of biomass removal but generally requires prior flocculation for high removal efficiencies. Sedimentation can be carried out with some species without flocculation, but removal efficiency is generally considered poor. Flocculation can be used to increase cell dimensions allowing improved sedimentation. If car­ried out in conjunction with flocculation, a sedimentation tank can provide a reliable solution for biomass recovery [69].

2.2.5.2 FLOTATION

Flotation was a method of harvesting considered in the 1960s [60][70] however the recoverability of biomass was generally found to be poor with a wide range of reagents tested. It has been reported that using dis­solved air flotation mixed algal species could be harvested up to a slurry of 6% total solids; using electro-flotation, which creates air bubbles through electrolysis which then attach to the algal cells, mixed algal species could be harvested up to a slurry of 5%, but this approached required a signifi­cant energy input; using dispersed air flotation which uses froth or foam to capture the algal cells resulted also in similar results [69]. Existing research indicates that flotation offers a quicker alternative to sedimen­tation following algal flocculation, but more energy is required and thus cost is higher whilst providing a final product with lower total solids content.

Our study uses algae oil to supply remaining demands in oil routes after the use of residues, waste and bioenergy crops. Because commercial scale algae growing and harvesting is currently still in development, we only include significant algae use from 2030 onwards. The approach to using algae in our work is based on a recent Ecofys study [30] on the worldwide potential of aquatic biomass. This study identified a number of different long-term feasible potentials for aquatic biomass. The most conservative scenario only contains algae oil from microalgae grown in open ponds on non-arable land filled with salt water. The total potential for algae oil from this technology was estimated at 90 EJ of oil. We envisage an algae cultivation system where the non-oil algae biomass are used to provide a nutrient loop where possible and the required energy for the cultivation and processing of the algae.

The maximum amount of algae oil actually used in the demand side scenario [1] is 21 EJ of oil in 2050. Based on the yields calculated in the Ecofys study, this amounts to approximately 300,000 km2 (30 million hect­ares) use of non-arable land. The 21 EJ oil use is about 25% of the 90 EJ algae oil potential identified in the most conservative scenario containing only algae oil from microalgae grown in open ponds on non-arable land filled with salt water. Therefore, the algae oil use in the demand side sce­nario fits comfortably within the potential identified in the Ecofys study, especially as further potential from algae cultivation in open water may be tapped due to future technological progress.

E. coli cells when placed under extremely low frequency (ELF) magnetic field sine wave of 30 pT at 9 Hz, exhibited a change in the conformational state of the genome, which was maximum at 4 x 108 cells mL-1 while there was no such response at lower cell densities of 3 x 105 cells mL-1. Other than cell density, time of exposure also affected genomic conformation. The change in the conformational state of the genome is considered to be dependent on DNA parameters, i. e. molecular weight and the number of proteins bound to the DNA [9]. Thus the ELF field which is close to the ion cyclotron resonance parameters for a medium weight ion might be influencing these factors that ultimately elicit response on the conforma­tion. It was also proposed that the possibility of a resonance fluorescence effect where recombination of fluorescing radicals may act as signals for intercellular communication and participate in the synchronization of gene expression. Weak, static magnetic fields (0-110 pT) are shown affecting DNA-protein conformations in E. coli. The analysis by Binhi et al. [51] represented a dose-response curve for the static magnetic field. The curve however is peculiar in having three prominent maxima unlike other dose — response curves in nature that usually follow rising or decaying exponen­tial functions. They explained this peculiarity in the context of the ion interference mechanism. No alteration in the profile of stress proteins of E. coli was observed by Nakasono et al. [52] on exposure to AC fields (7.8-14 mT, 5-100 Hz). In Saccharomyces cerevisiae no changes were observed under AC magnetic fields (10-300 mT, 50 Hz) in differential gene expression and protein profile that were determined using microar­ray and 2-D protein profile analysis, respectively [53]. But, Gao et al. [54] reported that strong magnetic fields (14.1 T) could lead to transcriptional up-regulation of 21 genes and down-regulation of 44 genes in a gram­negative anaerobic bacterium Shewanella oneidensis that did not show any significant effect on growth. In the anoxygenic photosynthetic bacte­rium, Rhodobacter sphaeroides, AC magnetic fields of 0.13-0.3 T induced a 5-fold increase in porphyrin synthesis, and enhanced expression of the enzyme 5-aminolevulinic acid dehydratase, while very strong DC fields (0.13-0.3 T) also induced synthesis of this enzyme predominantly at the magnetic North Pole. The effects are attributed to elevated gene expres­sion that ultimately resulted in increased porphyrin production [25].

Mitotic delay of 0.5 to 2 h was observed in a slime mold Physarum polycephalum in presence of ELF electromagnetic fields (45, 60 and 75 Hz) by Goodman et al. [44]. Removal of the mold from magnetic field recovered normal mitosis in 40 days.

Routine determination of algal dry cell weight (DCW) was obtained by measuring the optical density of chlorophyll at 680 nm (OD680) using a spectrophotometer (Shimadzu UV-265). The conversion of OD680 to DCW was accomplished by generating a dilution series for each species, recording the OD680, and then collecting samples onto pre-weighed cel­lulose acetate membranes (Pall Co., Port Washington, NY, USA), which were then dried in a vacuum oven (15 in. Hg., 60 oC) for 12 h before ob­taining the final weights. To avoid optical filtering effects, samples were diluted, if need be, such that the OD680 was always less than 1.8.

In order to achieve large-scale biodiesel production from microalgae, a cost effective cultivation system is of great significance. The cultivation systems include open and closed styles. The former, which simulates the growth environment in natural lakes, is just open-ponds characterized by simple and low cost structure and operations, low biomass concentration, and poor system stability. The closed culture systems are photobioreac­tors (PBR) of different configurations including tubular, flat plate, and column photobioreactors. Compared with open pond systems, the closed systems are usually more stable because it is easier to control the process conditions and maintain monoculture and allow higher cell density, but they have higher capital and operational costs. In both open and closed microalgae culture systems, light source and light intensity are critical to the performance of phototrophic growth of microalgae. With the develop­ment of optical trapping system, light delivery and lighting technologies, which improve the distribution and absorption of light, the advent of some new photobioreactors will improve the efficiency of photosynthesis [17]. In addition, gas-liquid mass transfer efficiency is another critical factor affecting CO2 utilization and hence the phototrophic growth. Cheng et al. [18] constructed a 10 L photobioreactor integrated with a hollow fiber membrane module which increased the gas bubbles retention time from 2 s to more than 20 s, increasing the CO2 fixation rate of Chlorella vulgaris from 80 to 260 mgL-1h-1.

As with any production process, algal biofuel will undoubtedly have an impact on the environment relating to land use, water use, atmospheric emissions and terrestrial/water emissions. One of the key aims of biofuel production is to produce a fuel with fewer environmental impacts than conventional fossil fuels [100]. The intensive processing of the biomass, however, could result in a fuel with greater environmental impacts.

When considering environmental impacts of a product, many factors are taken into account. One of the main impact categories which is con­sidered is the greenhouse gas emissions (GHG) in kg CO2 equivalent, ef­fectively the benchmark for how “green” a product is. In the best case, a biofuel can have a negative greenhouse gas emission in that during its pro­duction more carbon dioxide is taken up than is released during produc­tion and use of the fuel. Many studies have been carried out assessing the greenhouse gas emissions of various fuel types from different feedstocks. Recent studies which have investigated the production of algal biofuel have found that, under most circumstances, algal biofuels are likely to have a net positive greenhouse gas emissions [77, 87, 96]. This is in con­trast with many other biofuels produced from conventional first and sec­ond generation feedstocks which are produced uptaking more greenhouse gases than are emitted in the process [101-103]. A comparison of carbon dioxide emissions from algal biofuel and alternative feedstocks is shown in Table 9. The table shows the CO2 emissions per MJ of energy recov­ered as biofuel. The LCA method is included showing at which point the study stopped i. e., at fuel production (well to fuel) or at combustion (well to wheel). The data displayed in table 8 exhibits how poorly algal biofuel currently performs when compared to alternative feedstocks whether they are processed to bioethanol or biodiesel. One of the studies finds algal biodiesel to provide a negative GHG balance [95], nevertheless this is in contrast to the majority [77, 87, 96]. The different termination points of the study make comparison more difficult as predictably there are GHG emis­sions associated with the transport and combustion of the fuel.

The majority of greenhouse gases in algal biofuel production are emit­ted as a result of energy production. Clarens et al. [87] for example dem­onstrated that CO2 procurement demands 40% of total energy consump­tion and 30% of GHG emissions. Any electricity required will create GHG emissions at the point of generation. In their more recent study Clarens et al. [97] compared the greenhouse gas emissions of two scenarios: algal biodiesel with bioelectricity generated from residual biomass and just bio­electricity generated from the biomass. The results were compared with biodiesel and bioelectricity from canola and bioelectricity from switch — grass. The energy from algae scenarios both performed well with direct bioelectricity from algae producing the least GHG emissions. The process stream configuration greatly affects the energy requirements. The greater the number of processes (particularly those including lipid extraction and digestion) required more energy and thus also produced greater green­house gas emissions.

GHG emissions may be the most common impact category yet there are many others that also require consideration including eutrophication potential, global warming potential, land use and human toxicity. In their life-cycle analysis Lardon et al. [77] investigated the environmental im­pacts of their algal-biofuel best-case scenario (low N, wet processing) to alternative feedstocks (rapeseed, soybean, palm, diesel). In some areas the algal biofuel performed well (such as in land use and eutrophication) how­ever it did not compare well for the majority of categories, particularly for photochemical oxidation, ionizing radiation, marine toxicity, acidification and abiotic depletion. In the study published by Clarens et al. [87] a fewer number of categories were investigated but the results are similar for eu­trophication and land use, both of which are favourable in comparison to corn, canola and switchgrass. Clearly improvements need to be made to minimise the adverse impacts that would be caused by the production and combustion of algal biofuels. These impacts are unlikely to ever be non-existent but it is important that the concept can perform favourably in comparison to alternatives regarding environmental impacts.

The Highly Productive Case is an analytical model that was constructed to represent a system with greater biomass productivity (80 mg/L-d) and a higher neutral lipid fraction (30%) than the Experimental Case (which had productivity of 2 mg/L-d and lipid fraction of 2%). The Highly Produc­tive Case assumes the same basic production pathway as the Experimental Case, but it substitutes bioreactors for growth and an advanced floccula­tion technique in place of centrifugation. In addition, several modifica­tions are modeled to improve energy efficiency in the Highly Productive Case. In addition, it is assumed that there is no water loss from evapora­tion and 95% of the water used for cultivation was recycled. In this sense, the Highly Productive Case is an optimistic, but not wholly unreasonable, scenario for achieving low operating expense in commercial-scale algal biofuel production based on current technologies. The less optimistic as­sumption is the requirement of “full-price” inputs (such as nitrogen fertil­izer and carbon dioxide from ammonia production plants). The ability to achieve each of the specified conditions in the Highly Productive Case in
practice is assumed to be possible and the capital required to do so is not considered in this study. Each assumption used in the Highly Productive Case is compared with those from several literature sources in the “Dis­cussion” section, below.

The ability to utilize discounted inputs, such as waste forms of carbon, nitrogen, and phosphorus and cheap energy inputs, would further improve the return on investment for producing algal biofuels with respect to the Highly Productive Case [11,14,25-28]. The Highly Productive Case is not intended to represent the optimum scenario for algal biofuels nor is it presented as the final arbiter of the fuel’s prospects for success; rather, it is intended to serve as a useful benchmark. The optimum scenario might utilize discounted inputs, high productivity algal strains (e. g., genetically modified organisms), and improved growth, processing, and harvesting methods that might be developed in the future. Instead, the Highly Produc­tive Case models a similar production pathway as the Experimental Case, but with significantly higher fuel productivity and significantly more ef­ficient growth and processing methods.

From a geometric perspective, it is possible to compare two dividing cells in living systems with a Josephson junction of superconductivity [91]. The

Josephson junction may represent a gap junction between two nearby cells coupled via electromagnetic interactions, which provides a mechanism forthe transfer of correlated charged particles, electrons, and ions. The gap junctions serve to transmit electrical signals between adjacent cells with­out the need for mediation by a neurotransmitter or messenger substance [92]. Positive experimental results were attained in yeast cells by exam­ining their current-voltage characteristics and radiofrequency oscillation spectra during cell division [91].

Microalgae strains with potentially high lipid content (e. g., as determined by Nile red staining) need to be cultured to increase biomass and directly compared to each other in larger cultivation systems to assess their poten­tial as biodiesel feedstock. Initial tests of the most promising algae strains usually are carried out at laboratory-scale using culturing flasks and other vessels, such as hanging bags, under well-defined growth conditions. The test should follow a standard protocol over a certain growth period to allow direct comparisons between strains in terms of growth rate and lipid ac­cumulation (“lipid productivity”). It should be noted though that a standard assay does not take into consideration the potential of certain microalgae under carefully optimized conditions. An example of this assay is the fol­lowing that is routinely used by our laboratory to compare lipid productiv­ity: Pure (but not axenic) algae strains are cultured in F/2 medium (fresh or seawater) until near stationary growth occurs (less than 20% growth in 24 h as determined by cell counts). An inoculum of 5 mL of this culture is then used to start growth in 20 mL fresh F/2 medium exactly at 2 h after the start of the light cycle. The culture is then grown and monitored by cell counting for 7 days after which medium is replaced with nutrient-free water. Nutrient starvation is conducted after that for another 2 days of cultivation to test the potential for rapid TAG accumulation. In addition, biomass is collected at the end of the experiment for lipid content analysis. This assay is useful for screening of growth and lipid producing capacity of microalgae, leading to selection of potentially useful strains. The best candidate strains with po­tential for biodiesel production should then be used to optimize parameters for rapid growth, lipid induction, harvesting/dewatering and oil extraction. While most of these parameters are typically optimized under small-scale laboratory conditions, it seems advisable to move towards larger size out­door cultivation conditions as soon as possible, as these are typically quite different. Parameters, such as salinity, nutrient composition, pH and cell density can be controlled to some extent, but other factors such as tempera­ture, irradiation and the co-cultivation of other organisms are much harder to control under outdoor conditions.

In summary, it is advantageous to isolate and screen a large number of local microalgae strains and test these under mid-scale outdoor condi­tions as soon as possible to be close to conditions that maybe expected for large-scale cultivation. Figure 2 provides a step-by-step overview of how microalgae may be rapidly isolated and selected for larger scale biodiesel production.