Regarding agricultural nutrient use, we envisage closed nutrient loops where possible. In addition, we included in the demand side of the Ecofys Energy Scenario, to which this study is related [1]:

• All heat energy input required to produce N fertiliser for bioenergy cropping.

• All electricity required to produce hydrogen for N fertiliser for bioenergy cropping. This mechanism can also be used as storage for supply-driven renewable electricity sources (see [1]).

3.3.2.2 PROCESSING WATER USE

We assessed, from literature, expert opinions and our previous experience, that a closed loop approach for biofuel processing water is currently com­mercially available.

6.3.1 GROUP I: TREATMENTS INVOLVING MAGNETIC FIELD PREDOMINANCE

Experiments involving a predominant magnetic field have been conducted on a range of microorganisms that represent both prokaryotes (eubacteria, archaea) and eukaryotes (algae, fungi, protozoa).

6.3.1.1 GROWTH

Growth is a physiological response of an organism and a positive effect on growth indicates that some of the biosynthetic pathways are being stimu­lated. Erygin et al. [15] grew a gram-positive bacterium Bacillus mucilagi­nous in a magnetic field of ~0.26 T under different media compositions and compared it with unexposed control cultures. The magnetically treated liquid medium consisting of ferromagnetic salts showed rapid growth of the bacterium over control in 3 h. Similarly magnetically treated dry whey medium yielded three times higher cell count than the untreated medium. However, there was an overall increased response from the exposed dry whey illustrating how the culture medium composition may influence the effect of magnetic field.

Moore [17] studied five strains of bacteria and a yeast under a magnetic flux of 5-90 mT and reported maximum stimulation of growth at 15 mT (at 0.3 Hz) and maximum inhibition at 30 mT. Experiments with varying time especially using oscillating magnetic fields have uncovered new ef­fects related to resonant phenomena in the living systems. The biostimula­tion of a denitrifying gramnegative bacterium Pseudomonas stutzeri by a magnetic field of 0.6-1.3 mT pulses via inductively coupled Helmholtz coils for 8-10 h resulted in a proliferation of biomass that was 10-30% more than the control [18].

Other than the medium conditions, magnetic flux and type of magnetic field, the exposure time is another major factor that governs the intensity of response. Justo et al. [8] observed that the growth of Escherichia coli could be stimulated or inhibited by exposure to an oscillating 100 mT extremely low frequency (ELF) magnetic field for 6.5 h. Exposed cells had 100 times greater viability than unexposed cells, however the viability varied with duration of exposure. It was suggested that the effect was a result of magnetic field driven alteration of membrane permeability and availability of ions in the culture medium.

Research groups in Japan and China have focused on investigating ways to improve the cultivation of the cyanobacterium Spirulina platensis for production of nutraceuticals using permanent magnetic fields. Hirano et al. [26] reported a significantly higher specific growth rate of 0.22 d-1 in S. platensis exposed to 10 mT magnetic field when compared to 0.14 d-1 for untreated culture. The growth of S. platensis was maximum when it was cultured phototrophically at lower light intensities; but did not show improvement under heterotrophic conditions.

Magnetic field induced growth stimulation in S. platensis has also been reported by Li et al. [27]. They observed a 47% increase in dry biomass on the sixth day of cultivation, and a 22% increase over control by day eight under the exposure of a 250 mT homogeneous magnetic field from a Helmholtz coil.

Chlorella vulgaris is another algal strain of interest for its nutraceutical value and is a promising producer of starch-glucose. This microalga can yield starch to the tune of 60 t ha-1 yr-1 which is 7.7 times more than that of traditional corn [50]. Takahashi et al. [31] used magnetic flux densities of 6-58 mT for cultivating Chlorella sp. and obtained facilitative growth at 40 mT. The specific growth rate of Chlorella vulgaris almost doubled from

0. 07 to 0.12 d-1 under magnetic field generated using a dual-yoke electro­magnet, which concentrates a magnetic field into a small cross-sectional area [30].

The static magnetic field strengths ranging from 0 to 230 mT on Du — naliella salina were used by Yamaoka et al. [32]. They observed an im­provement in growth rate that peaked at 10 mT with the addition of 1 mg L-1 of Fe-EDTA. A ~0.26 T magnetic field exposure using different growth media for the yeast Saccharomyces fragilis showed that rapid growth (27- 36% over the control in 3 h) occurred on magnetic treatment when a dry whey nutrient medium was used, but it turned inhibitory on using a liquid nutrient medium [15]. On the other hand Fiedler et al. [36] used an oscil­lating magnetic field generated by a Helmholtz coil via inductive coupling to produce 0.28-12 mT magnetic field at 50 Hz for 9 h to treat S. cerevisi — ae. They observed a maximum growth of 8 g L-1 of biomass under 0.5 mT magnetic field exposure and 6.8 g L-1 of biomass for the cells untreated.

While we are not equipped to do a formal life cycle analysis and feel it would be premature given the improvements needed in resin technology, nevertheless a comparison of the proposed resin-based processing to current approaches seems in order. Figure 5 presents a general schematic for current algal processing schemes derived on published evaluations [5,29]. For the most part, the cost of growing algae would be the same. The use of resins could impact growth in terms of the need for nitrogen starvation or growth of monocultures as discussed below. Primarily, our one-step harvest to biodiesel approach would impact the most expensive steps in processing algae to biofuel, namely the harvesting and extraction steps.

Currently harvesting typically requires both pumping the algal suspen­sion and some treatment such as centrifugation, flocculation or dissolved air flotation. Centrifugation and dissolved air flotation both have relatively large capital costs and substantial electricity. Where resins could make a difference is by eliminating the need for pumping water. While we have used resin beads for comparisons, we do not feel this is the best way to deploy them for harvesting. Resin beads are porous with a large internal volume that can trap water. The pores are typically 10-30 nm in diameter whereas the algae are on the order of 2-3 pm in diameter so they cannot enter the resin. Our measurements show that after the bulk water is re­moved, about 100 mg of water/gram resin is retained either inside the resin or entrained between the beads. A better alternative is to use thin films of resin where the internal volume is low compared to the surface area. We have proposed elsewhere a belt harvester type approach where a belt moves from the pond, where it picks up the algae to a vat which, in this case would contain sulfuric acid/methanol [25]. In that study we estimated that a bristled belt 1 m wide x 7.5 m long could collect 3 kg algae per one complete turn of the belt. Other modes are possible such as mesh bags con­taining nonporous resin-coated particles that float or are semisubmerged.

Once the algae is harvested and eluted, the conversion to biodiesel is direct. There is no need for lysing, further drying and solvent extraction, steps that are considered quite expensive [5]. Hexane extraction requires recovery of the hexane and a certain amount will be lost to the environ­ment. Furthermore, hexane only recovers the neutral lipids (mainly TAG). Our experience at measuring TAG in pond-grown algae is that TAG levels rarely surpass 5% DCW and we have never seen them greater than 12% DCW. One of the advantages of this approach is that FAME is generated from both polar and neutral lipids thereby increasing the yield from the

A Conventional

В Resin-based

algae. Our data suggest improvements of up to 4-fold were possible with an alga (KAS 603) that made 10% TAG.

The improved yield could affect several aspects of the algal growth process. Firstly, our data suggested that there was not so much to be gained by nitrogen starvation. Both starved and unstarved algae contained similar amounts of total lipid. Admittedly, the spectrum of fatty acids was differ­ent and this might be a factor. Secondly, resin-based conversion of total lipid to biodiesel might make it less important for growers to maintain a
monoculture of high TAG-producing algae. Of course, high TAG produc­tion is a benefit but it is difficult to maintain monocultures in open ponds or in potentially other attractive scenarios where algae are grown in con­junction with wastewater treatment plants. Finally production of biodiesel onsite eliminates the transportation cost of moving algal oil to a biodiesel plant. While the biodiesel must be recovered from the sulfuric acid metha­nol, we believe this can be done using a low solvent or even solventless hollow fiber extraction system. In commercial biodiesel plants, the glyc­erol separates from the biodiesel layer and the separation is facilitated by water. This may be possible as well but more study is needed to determine how best to fractionate the spent sulfuric acid/ methanol mixture. There will be many cellular components in the final sulfuric acid/methanol solu­tion, some of which may be valuable as side products.

XIAODAN WU, RONGSHENG RUAN, ZHENYI DU, and YUHUAN LIU

1.1 INTRODUCTION

In recent years, increasing consumption of conventional energy has caused serious concerns about energy security and environmental degradation. Therefore, renewable, non-polluting biomass energy has been receiving more and more attention from both the academic community and indus­tries. Biomass derived from photosynthesis includes a variety of organ­isms, such as plants, animals and microbes. As photosynthetic microor­ganisms, microalgae can use and therefore remove nitrogen, phosphorus in wastewater, sequester CO2 in the air, and synthesize lipids which can be converted into biodiesel. The declining supply of conventional fossil fuel and concern about global warming make microalgae-based biodiesel a very promising alternative. Although the potential and advantages of microalgae-based biodiesel over conventional biodiesel have been well recognized [1-3], broad commercialization of microalgae biodiesel has not yet to be realized, chiefly because of the techno-economic constraints, particularly in the areas of mass cultivation and downstream processing. The objectives of the present paper are to review recent development in microalgae production, especially in high density cultivation and down­stream processing, and identify technological bottlenecks and strategies for further development.

With water becoming a scarce commodity, intensive use of water for bio­energy cannot be considered sustainable if water extraction is affecting ag­riculture, domestic use or causing environmental impacts. Being an aquat­ic species, algae require more water than terrestrial bio-energy plants and when cultivated in open ponds there are great water losses mainly through evaporation. According to Williams and Laurens [93] the dissociation of one mol of water occurs for every mol of CO2 required in the photosyn­thetic process. In their study of water use in algal cultivation Murphy and Allen [94] calculate that 33.2 m3/m2 of water per year is required to culti­vate algae in a raceway pond in the United States. It is possible to recycle much of the water that is drained from the ponds during harvesting but there will be losses in harvesting the algae; freshwater must therefore be sourced. In the same study it was reported that the management of the water will require seven times the amount of energy that can be produced from biodiesel extracted from the algae [94]. The majority of countries around the world are becoming increasingly water stressed and therefore using extra freshwater in biofuel production is not sustainable. The use of wastewater as an alternative to freshwater provides an ideal solution how­ever freshwater would still be necessary for downstream processing of the biomass or for dilution of highly concentrated wastewater.

Many countries including the European Union (EU) have adopted policies on certain percentage of renewable energy use for transport and other rel­evant sectors. In December 2008, the EU signed a directive that requires 10% of member to come from renewable sources (biofuels, hydrogen and green el policy towards mitigation of climate change effect and global warming. The EU directive also obliges the bloc to ensure that biofuels offer at least 35% carbon emission savings compared to fossil fuels and the figure should rise to 50% in 2017 and debates among governments, policymakers, scientists and environmentalists as currently most commer­cially produced biofuels are derived from sources that compete with or belong to feedstock for human and animal consumption.

In terms of greenhouse gas emission, the biofuels produced from microalgae is generally carbon neutral. The CO2 emitted from burning biofuel is assumed to be neutral as the carbon was taken out of the atmosphere when the algae biomass grew. Therefore, biofuels from microalgae do not add new carbon to the atmosphere. Biofuels can be a viable alternative to fossil fuels on short and medium terms. Additionally, advanced biofuels made from residues or waste have the potential to reduce CO2 emissions with 90% compared to petrol/diesel.

While many years of research and development still lie ahead, if suc­cessful, algae-based fuels can help meet the world’s growing demand for transportation fuel while reducing greenhouse gas emissions. However, a number of challenges remain before algae can be used for mainstream commercial applications as the uncertainty of cost constitutes the biggest obstacle. There is no doubt that research work on microalgae is still in primary stages. Currently, it is not clear that what kinds or families of algae would be most appropriate in order to produce commercially viable biofuels. Researchers are currently working on appropriate commercial cultivation processes of algae biomasses. At this point in time, there is no definitive answer to an open question if it is better to grow algae in photobioreactor system or open air (pond) system. As algae are micro-or­ganisms of a size ten times smaller than human hair, it is a great challenge to harvest them. At present, microalgae harvestings are based on either centrifugation or chemical flocculation, which push all the microalgae to­gether, but these processes associated with high cost [16-23].

Biodiesel or bioethanol production from algae biomass cannot be com­mercially viable unless by-products are optimally utilised. As mentioned earlier, the lipid or the oil part is around 30% of the total algae biomass and the remaining 70% is currently wasted which can be used as nutrients, pharmaceuticals, animal feed or bio-based products. The use of lipid as well as all by-products will allow exploring the full potential of microal­gae towards sustainable environment and economy. At present, 70 -90% of the energy put into harvesting microalgae for fuel usually gets used into extracting the lipids (oil) they produce under current factory designs. It is obvious that new technologies are needed for reducing huge energy losses [13, 23-27].

Microalgae have immense potentials for biofuels production. How­ever, these potentials largely depend on utilisation of technology, input feedstock (CO2, wastewater, saltwater, natural light), barren lands and marine environment. Based on energy content, available technology, land, it is hard to overemphasize that biofuels are a realistic short-term, but definitely not a long-term and large scale solution to energy needs and environmental challenges. Microalgae can be temporary sources of energy, and with the appropriate growth protocols they may address some of the concerns raised by the use of first and second generation biofuels.

Magnetic fields cause oxidative stress in organisms by altering energy lev­els and spin orientation of electrons and concentration and lifetime of free radicals, which change the relative probabilities of recombination of other interactions with possible biological consequences [67]. Oxidative stress due to the radical pair mechanism becomes applicable around 1 mT which can be common in industrial or laboratory settings, while the geomag­netic field intensity stays below 0.07 mT. Studies with Chlorella vulgaris demonstrated that hydroxyl ions increase in magnetically treated medium suggesting alteration of free radical levels in the medium that might hy — peractivate antioxidant defense system of the organism. This situation also affects the membrane permeability and ion transport process and might be responsible for the acceleration of chlorophyll excitation by the light [30].

The developments in the biodiesel industry have progressed dramatically in recent years. Developed countries have set priorities on biodiesel fu­els for the transport and mechanical industry and established a Biodiesel Board for policy making and development. The production of biodiesel in the EU has been increasing from 1.9 million tons in 2004 to 3.2 million tons in 2005 and to 4.9 million tons in 2006 [5]. In 2011, biodiesel produc­tion rose to 22.117 million tones as reported by the European Biodiesel Board (EBB); the leading biodiesel producing countries in the area include Germany, France and Spain [6]. The United States is also developing bio­diesel applications in many different industries. According to the US Na-

tional Biodiesel Board, biodiesel production increased from 0.016 million tons in 1999 to 0.787 million tons in 2004 [7]. Production and sales were estimated to have tripled from 2004 to 2005 and to have reached 21.73 million tons of fuel in 2008 and more than 31.5 million tons in 2011, as reported by The U. S. National Biodiesel Board (TUSNBB) [8]. To date, research on biodiesel production from microalgae is enthusiastically at­tempted globally.

In comparison with other sources (e. g., animal fat, oleaginous grain crops and oil palm), there are remarkable advantages of biodiesel from mi­croalgae as an alternative energy source for the future. Advantages include the following: (i) areal growth rate and oil productivity of microalgae per unit of land use are much higher than those of other biofuel crops; (ii) algae grow in a wide range of environments. Fresh, brackish and saline waters are ideal environments for growth of different algae species. Even in municipal and other types of wastewater, algae grow well by using in­organic (NH4+, NO3-, PO43-) as well as organic sources of nutrients [9]; (iii) microalgae absorb CO2 photosynthetically and convert it into chemi­cal energy and biomass. The removal of CO2 from the atmosphere (and possibly industrial flue gases) may play an important role in global warm­ing mitigation by replacing fossil fuel emissions [9,10]. Producing 100 tonnes of algal biomass fixes roughly 183 tonnes of carbon dioxide from the atmosphere [4]; (iv) microalgae can provide raw materials for different types of fuels such as biodiesel, ethanol, hydrogen and/or methane which are rapidly biodegradable and may perform more efficiently than fossil fu­els [11]; (v) products extracted from algal biomass can be used as sources for organic fertilizers or high value products, such as omega-3 fatty ac­ids, sterols, carotenoids and other pigments and antioxidants, and could be amenable to a zero waste biorefinery concept [9]. Therefore, microalgae have been regarded as possibly the only route to sustainable displacement of high proportions of fossil oil consumption.

Intracellular microalgae lipids can be extracted by a variety of methods, such as mechanical crushing extraction, chemical extraction, enzymatic extraction, supercritical carbon dioxide (SCCO2) extraction [46], micro­wave extraction [47], etc. Microalgae lipids in the form of triglycerides or fatty acids can be converted to biodiesel through transesterification/ (esterification for fatty acids) reactions after the extraction [48]. In order to achieve efficient reaction, the choice of catalyst is very important. The traditional liquid acid and alkali catalyst are called homogeneous catalysts because they act in the same liquid phase as the reaction mixture. Due to their simple usage and less time required for lipids conversion, the ho­mogeneous catalysts dominate the biodiesel industry. However, the trans­esterification catalyzed by homogeneous catalysts needs high purity feed­stock and complicated downstream processing [49], so high efficiency and low pollution catalysts such as solid acid catalysts, solid alkali catalysts, enzyme catalyst, supercritical catalyst systems and ionic liquid catalysts are receiving increasing attentions. Krohn et al. [50] studied the catalytic process using supercritical methanol and porous titania microspheres in a fixed bed reactor to catalyze the simultaneous transesterification and esterification of triglycerides and free fatty acids to biodiesel. The process was able to reach conversion efficiencies of up to 85%. Patil et al. [51] re­ported a process involving simultaneous extraction and transesterification of wet algal biomass containing about 90% of water under supercritical methanol conditions.

The proposed concept utilises a variety of waste streams from industry thus saving energy and environmental impacts by avoiding manufacture of raw materials. Furthermore as a method of wastewater treatment, en­ergy and associated impacts will be saved by avoiding alternative meth­ods of treatment. The energy recovered through anaerobic digestion or combustion can be returned to the system, powering the units which re­quire an energy source (paddlewheel or centrifugation). The waste heat can be used to dry the biomass if required or alternatively used to heat the ponds if the temperature falls below optimal conditions. The residual waste from the energy recovery system can be fed back into the treat­ment ponds supplying additional nutrients if required or alternatively sold as a fertiliser.