The PFROI is equivalent to the QA 2nd O EROI and is calculated using Equation (14). This relation serves as a standard way to compare energy and cost analyses at a systems level. By doing so, the energetic profitabil­ity of an energy system (which is the most important metric for researchers interested in global energy production and consumption or thermodynam­ics of energy systems) can be compared with the financial profitability of an energy system (which is most important to businesses and investors).

The cost of growing algae was calculated for the Experimental Case by applying electricity and material prices, yielding a total cost of growth of $105.2/kLp. With 2.1 g of bio-oil produced from each kL of processed

URE 2: The EROI and PFROI for the Experimental Case and the Highly Productive Case decline as more inputs are considered. The curves are presented for illustration only, as the curve shapes are unknown.

volume, these cultivation costs are $40,000/L of bio-oil ($150,000/gal). The Highly Productive Case data results in a total cultivation cost of $0.42/kLp, which is equivalent to $0.42/kg of algae or $1.6/L of bio-oil ($6.1/gal) based on the bio-oil productivity calculated above (210 g bio — oil/kLp). The combined cost of processing and refining was calculated to be $7.71/kLp and $0.13/kLp for the Experimental and Highly Productive Cases, respectively (cf. Tables 1A and 2A). Based on the resulting bio-oil productivities, these values correspond to $2900/L of bio-oil ($11,000/gal)
and $0.5/L of bio-oil ($1.9/gal) for these cases, respectively. Davis et al. present a comprehensive techno-economic analysis of a similar production system (including capital costs) and determined that operating costs for both open-pond and enclosed bioreactor settings would be near $1.3/L of bio-oil ($5/gal) [16]. This result is similar to the total operating cost of the Highly Productive Case ($2.1/L of bio-oil, $7.99/gal). In the Experimental Case, 2.1 g of bio-oil were produced per kLp (0.0026 L/kLp) and 41.6 g of methane were produced per kLp. Assuming market prices of $0.66/L of bio-oil ($0.66/L, $0.83/kg) and $4/MMBtu of methane ($0.21/kg) yields revenues of $0.0017/kLp for bio-oil and $0.0087/kLp for methane in the Experimental Case (yielding $0.010/kLp of total revenue). In the Highly Productive Case, 210 g (0.26 L) of bio-oil and 150 g of methane are pro­duced for each kLp, resulting in $0.17/kLp of bio-oil revenue and $0.03/ kLp of methane revenue. Until 2012, a production subsidy of $0.13/L was provided for corn ethanol in the United States, and if an equal subsidy was provided for algal fuels, the production plant would gain incremental income of $0.0004/kLp for the Experimental Case and $0.035/kLp for the Highly Productive Case.

The partial financial returns on investment (PFROI) are calculated from Equation (14) for the Experimental Case and the Highly Productive Case to be 9.2 * 10-5 and 0.37, respectively. The challenge in obtaining a PFROI greater than 1 is growing, processing, and refining high-yield biomass cheaply, especially since many of the costs scale directly with biomass productivity (e. g., nutrient costs increase as biomass productivity increases). The overall FROI would be lower than the PFROI as capital, labor, and distribution costs will be significant expenses, which are not included in the PFROI. For example, Lundquist et al. and Davis et al. provide analyses for capital costs of similar production systems and dem­onstrate that capital costs might contribute roughly 50% of the total cost for open-pond systems (this fraction increases substantially for bioreac­tors) [16,52]. Figure 2 illustrates the relationships between the EROI, QA EROI, and PFROI with respect to the number of inputs that are considered in the analysis, and is based on the work of Henshaw, King, and Zarnikau in relating EROI to full business costs, or cash flows [53]. For a given biofuel output, as more inputs are included in the calculations, the return on investment values decrease.

The application of exogenous electromagnetic influences has been used for various commercial applications and an overview is given in Table 2.

The electric field pulses, or electroporation, have been traditionally implemented in metabolic engineering for gene transformation. Direct electroporation of a cyanobacterium Synechococcus elongates, introduced the enzyme Clostridial hydrogenase, which may lead to the development of a variety of hydrogenases for hydrogen production, coupled to pho­tosynthesis in cyanobacteria for bioenergy production [186]. In addition to membrane-permeabilizing effects, it can also induce biochemical and physiological changes in plant protoplasts, such as stimulating protein and DNA synthesis, and cell division and differentiation [187]. Alternatively, electroporation can also be used as a process for cell membrane modifi­cation for enhanced oil/lipid extraction from microalgae for biodiesel. A preliminary study found 20% increase in oil yield, shorter extraction time, and 2/3 less solvent used without affecting the composition of extracted fatty acids compared to chemical solvent alone [33].

Although most electrochemical and electromagnetic effects mentioned thus far have been focused on biological responses, an integrated biorefin­ing system also requires process engineering technologies for harvesting algae for instance. An electrochemical process using direct electric cur­rent, called electroflocculation, has a long history as a wastewater treat­ment technology for solid/liquid, and liquid/liquid separation [188]. This technology combines the use of a sacrificial electrode that dissolves to coagulate suspended particles (electrocoagulation) along with the use of electrolysis, which produces H2 microbubbles that float the aggregates or flocs to the surface (electroflotation) for easy removal from the water. Electroflocculation is a promising technology for harvesting microalgae biomass since it has several advantages over other conventional processes. The efficiency of particle/biomass separation in electroflocculation is over 90% and this technology does not require moving parts, and consumes relatively little energy (0.3 kWh m-3) with substantially lower capital costs [189]. In fact, a more recent study shows a 99.5% removal of total sus­pended solids (TSS) and chlorophyll a (algae) by applying 0.55 kWh m-3 for 15 minutes [190].

Bioelectrochemical denitrification is a novel technology being used for the treatment of ammonium and nitrate-containing wastewater by means of denitrifying bacteria and hydrogen gas produced on the cathode by the electrolysis of water. The denitrifying microorganisms are usually immo­bilized as a biofilm on graphite or a stainless steel cathode. A nitrate re­moval efficiency of 98% was observed at 20 mA when phosphate was used as a buffer. The studies suggested that the application of bioelectroreactors could be used for reduction and oxidation treatments of ammonium and nitrate-containing wastewaters [191-193].

In many cases, real-time monitoring of cultures is critical for produc­tive and efficient cultivation/fermentation in which the optical density, pH, and dissolved gas levels may not elucidate the underlying bioprocesses occurring, especially when evaluating electrochemical or electromagnetic interactions. Pulsed Amplitude Modulation Fluorometry or PAM fluorom — etry is a special method for measuring fluorescence from photosynthetic organisms for real-time culture monitoring of the photosynthetic appara­tus. It uses the characteristics of the fluorescence emitted by chlorophyll a as a probe for the biophysics and biochemical events occurring in the electron transport chain of Photosystem I & II. These measurements are a unique indicator of photosynthesis and provide information about the maximum photosynthetic efficiency (by a dark-adapted sample), the effec­tive photosynthetic efficiency (under constant illumination), and the non­photochemical quenching (heat dissipation). These parameters indicate what fraction of the photon energy absorbed by the organism is used for photochemistry, dissipated as heat, and re-emitted as fluorescence [194]. Papazi and his colleagues found that PAM Fluorometry in conjunction with traditional biomass analysis was able to show how extremely high CO2 concentrations impacted the photosynthetic apparatus, which stimu­lated intense biomass production in the microalgae, Chlorella minutissima [195].

The use of the biophotonic method of delayed luminescence (DL) has been used for quality control applications with fruits and vege s. A study with tomato fruits revealed marked changes due to different harvesting maturities. It was found that tomatoes exhibited DL measurements related to color and respiration as well as significant differences in soluble solids content and dry matter percentage. Therefore, DL values are directly re­lated to tomato harvest maturity. Qualitative traits can depend on harvest maturity, thus suggesting that delayed luminescence could be used as a nondestructive indicator of fruit quality [196].

In addition to fluorescent measurements, the fast, non-invasive mea­surement of biological cells by dielectric spectroscopy, or impedance spectroscopy, is currently being utilized to determine cellular parameters, such as living cell volume, cell number distribution over cell cycle phase, cell length, internal structure, complex permittivity, and intracellular and extracellular media and morphological factors. The electrical and

morphological properties of the cell membrane are assumed to represent sensitive parameters of the cellular state [197]. It has been demonstrated to be a powerful method for dielectric monitoring of biomass and cell growth in ethanol fermentation and the extension of the scanning dielectric micro­scope is a promising tool for dielectric imaging of biological cells [198]. The real-time monitoring of yeast cell division by measuring the dielectric dispersion can enable to tracking of cell cycle progression using an elec­tromagnetic induction method [199]. Recently, online monitoring of lipid storage in microorganisms (yeasts) was conducted which found that us­ing dielectric spectroscopy data, the change in capacitance divided by the characteristic frequency being used showed a clear shift from the growth phase to the lipid accumulation phase, which could be of use for technical control of intracellular biopolymer or oil accumulation, as well as enzyme overproduction [200]. Moreover, it has been established that there exists a connection between D. L. and impedance spectroscopic parameters, which explore related structures and mechanisms in living samples [201]. By applying the knowledge, gained from biophotonic and bioelectromagnet­ic experiments, it may be possible to detect, interpret and interact with the endogenous coherent electromagnetic signals that are correlated with regulation, communication, and organization of biological systems since oscillation dynamics are of essential importance in intercellular and intra­cellular signal transmission and cellular differentiation [70]. These signals may initially give us real-time insight into the internal dynamics of an organism or culture, which may precede the physically/chemically observ­able events.

Induction of specific cellular response to biophotonic signals could perhaps be achieved to stimulate a desired biological effect such as en­hancement of lipid or enzyme synthesis or metabolite modulation using electromagnetic fields instead of an external stress or a biochemical ini­tiator. Electromagnetic bioprocesses such as electroflocculation and elec­troporation can be used for algal harvesting and biomass processing. The use of static and oscillating electromagnetic fields has a potential for the enhancement of cell proliferation, metabolite production and cell cultiva­tion for biomass production. After extraction, fermentation of the algae feedstock, using applied electric field parameters, can be designed for en­hanced substrate utilization and higher ethanol/butanol yields. Any residual biomass may then be used for enhanced production of methane from an­aerobic digestion using specific frequencies of microwaves reported by Banik et al. [5], who showed how the EM exposure parameters could be used for potential bioenergy/biofuel applications.

The application of electromagnetic coupling to electrochemical bio­logical pathways, which have been studied and commercialized for bio­medical applications can be introduced into bioengineering. Here inves­tigations into the electrochemical impedance properties for triggering biochemical cascades of desired signaling pathways in microorganisms for bioenergy applications deserve significant attention. The application of exogenous EMF influences may synergistically couple with endogenous electric fields for enhancing directed mass transport in cells. It is conceiv­able that any cell could be stimulated, inhibited, or made to exhibit passive response, depending upon the appropriate choice of frequencies and am­plitudes of the excitation signals employed [62]. The induction of mitosis for cell proliferation, as well as the stimulation of enzymatic pathways as­sociated with energy metabolism and storage such as lipid accumulation, needs modeling and more experimentation. Such electrochemical process­es may also be relevant for accelerating enzymes, such as Rubisco, in the carboxylation pathway of photosynthesis to enhance specific binding of CO2 and limiting photorespiration to enhance overall system efficiency in microalgae or plants. The greatest challenge may be the evaluation of the proper dosimetry for modulation of the desired biochemical cascade [1].

The introduction of the complex topology of multipolar electromagnet­ic fields may provide an enhanced coupling effect to complex, interdepen­dent biological systems. Such systems may be tailored to uniquely control endogenous electromagnetic processes and communication for cellular functioning and organization. Furthermore, the bioproducts, generated by engineered multipolar hybrid biosystems have additional properties. For example biofuel/bioenergy production processes potentially can have higher productivities through better substrate utilization and conversion and shorter processing times.

The use of electrochemical/electromagnetic triggering of specific metabolic pathways could be coupled with biophotonic analysis, where rapid screening and fine tuning of a desired effect could be devised. Such bioelectromagnetic and biophotonic monitoring could also be of significant

Algae

Harvesting

m ethane production

КО со

FIGURE 7: Bioengineering of algae cultivation.

interest to metabolic and genetic engineering, by incorporating and corre­lating electrochemical and endogenous electromagnetic signals with gene expression and enzymatic activity. The pervasive utilization of water in the cultivation of microorganisms particularly with algae, suggests pos­sible application of the principles discovered in ultra-high dilution and activation studies to enhance and modulate biological responses. Water used in the growth medium for cultivation may be imprinted by various methods (electromagnetic information transfer probably being the most convenient) with specific information on relevant organic and inorganic nutrients as well as biochemical growth promoters to enhance growth characteristics, while decreasing demand for the potentially large amounts of the donor substance.

The combination of these separate disciplines, could blossom into a new integrative bioengineering approach that incorporates the diverse specializations of molecular biology, biochemistry, electrochemistry, bio­physics, and quantum physics that could open up significant biotechno­logical progress of engineering of living systems for bioprocessing, bio­conversion, biofuel and bioenergy applications (Figures 6 and 7).

6.6 CONCLUSIONS

Traditional cultivation and manipulation of biological systems have con­sisted of natural selection and genetic engineering modalities. Recently metabolic engineering and synthetic biology are gaining wide attention from the scientific community due to their immense potential in altering the metabolism in living systems especially microbes for medical, agri­cultural, industrial and environmental applications. However, genetic ma­nipulation of microbes and living systems for agricultural and environ­mental applications may affect the ecosystem adversely as the changes in the species are permanent and inherited. In case of bioelectromagnetic stimulation, system reacts more in a transient fashion. The changes even if inherited are not sustained by the species for long thus they might be safer over genetic manipulation.

This review provided a broad spectrum of potentially useful bioef­fects on microorganisms that are currently or potentially valuable in biotechnology and bioenergy. At this point it is difficult to ascertain ex­actly how economically feasible these emerging methods and potential technologies will be due to a variety of unknown factors from the nature and scalability of the bioeffects to the electronic design and efficiency for large-scale implementation. However, it is the aim to stimulate interest in the field and invite scientists with new ideas into the long standing disci­pline of bioelectromagnetics that modern biology is only recently starting to understand. In the new horizon of biologically derived fuels and mate­rials, advancements in the area of biostimulation could impact the direc­tion of biotechnology towards an energetic approach that may boost the potential for emerging biotechnologies such as microalgae based biofuel and biomass production.

Biofuels, bioenergy and carbon capture are considered to be the cur­rent priorities for the entire global community. The International Energy Agency (IEA) has reported that the world’s primary energy need is pro­jected to grow by 55% between 2005 and 2030, at an average annual rate of 1.8% per year. Fossil fuels are the main source of primary energy and if the governments around the world stick to current policies, the world will need almost 60% more energy in 2030 than today. Transportation is one of the fastest growing sectors using 27% of the primary energy. At the pres­ent staggering rates of consumption, the world’s fossil oil reserve will be exhausted in less than 45 years. Considering the negative impacts of utiliz­ing fossil fuel energy sources, many countries have already mandated the use of biofuels and set the targets to replace significant quantities of fossil derived fuels. Second and third generation biofuels such as lignocellulosic ethanol and algae biofuels are considered to be the viable alternatives as they do not compete with food needs. Bioelectromagnetic stimulation of microbes particularly with microalgae provides a new extended domain of disciplines and methodologies for cultivation, harvesting and processing of biomass for production of biofuels, bioenergy and added value bio­products. Though this technology is promising, lots of research efforts are needed in future to exploit its commercial potential for biotechnology and biofuel applications.

Cultivation of algal biomass could provide a method of carbon mitiga­tion through CO2 uptake from flue gases during photosynthesis. Provid­ing algae can utilise industrial gases, there is the potential to remove CO2, which would otherwise be emitted. The mitigation of CO2 from flue gas using algae would be ideal in an industrial scenario, as targets for reducing greenhouse gas emissions are becoming tighter [44]. The improvement of biomass yields by introducing a concentrated source of CO2 has been reported [45, 46], however, there are many barriers yet to overcome; for example concentration of CO2 in flue gas may be too high for many strains of algae resulting in toxicity, and/or the presence of other toxins in the gas may adversely affect productivity, and/or gas transport cost to algal biomass growth reactors or ponds may be unviable. Nevertheless, as men­tioned, there is certainly potential for flue gases to play a part in an algal biomass cultivation system.

The atmosphere provides a CO2 concentration of0.038% for the growth of algae; theoretically, with a higher concentration available, higher pro­ductivity is possible [47]. Early studies found Chlorella sp. to be highly suitable for cultivation in flue gases due to its capacity to be grown with the injection of gas containing a CO2 concentration of 15% [48], a concen­tration similar to that of most flue gases [46]. Experimentation conducted within the US aquatic species programme [15] using flue gases as a source of CO2 indicated that local strains of algae dominated with a high CO2 use efficiency. Single algal biomass productivity rates as high as 50 g/m2/ day were recorded, although attempts to achieve consistently high produc­tivity rates failed during a long-term experiment for one year, provably due to low ambient temperatures [15]. In 2002, research was conducted

by the National Renewable Energy Laboratory (NREL) and the US De­partment of Agriculture investigating uptake of CO2 from synthetic and flue gas sources and its commercial and environmental viability. The technical feasibility and economic viability of integrating a micro-algal cultivation system with a coal fired power plant was investigated [49], using a bench scale system as a test rig. An artificial flue gas (12% CO2; 5.5% O2; 423 ppm SO2; 124 ppm NOx) based on the composition of a North Dakota power sta­tion boiler was produced and sparged into a bio-reactor tank. Two strains of algae were cultivated, Monoraphidium and Nannochloropsis, both of which grew successfully under the administered conditions. It was reported that growth rates of the microalgae varied between 15 to 25 g/m2/day and con­tained 41% protein, 26% lipid and 33% carbohydrate [49].

Research using real flue gases for CO2 uptake and cultivation of algal biomass has also been conducted [45, 46, 50, 51]. For example, Chlorella sp. was cultivated using a photobioreactor system approach and the pro­ductivity of Chlorella sp. was investigated in presence of a flue gas (6-8% CO2) from a natural gas boiler and in presence of a control gas, which resulted in higher productivity in the flue than in the control gas, of 22.8 ± 5.3 g/m2/day [52]. As a result it was suggested that 50% of the flue gas could be decarbonised using that system [51]. Similar studies conducted with Chlorella vulgaris using a photobioreactor system approach and flue gas from a municipal waste incinerator indicated that this strain was toler­ant to a concentration of 11% (v/v) CO2 as well as to the flue gas, with a higher biomass productivity in the flue [46]. Both studies suggest that the presence of potential contaminants in the flue had little adverse impact upon the algae. Examples of various strains of algae cultivated with the addition of CO2 with their productivity rates are summarized in Table 2. Existing research indicates that an improved growth of algal biomass has been obtained using artificial and flue gases with CO2 concentration up to approximately 12% (Table 2). Above this concentration it appears that productivity is reduced, most likely due to acidity caused by the high CO2 levels. It is suggested that, although most strains would benefit from an increased concentration of CO2, testing is required to identify optimal CO2 concentrations as this appears to vary between strains. Similarly only a limited number of flue gas sources have been investigated; if the concept is likely to be taken up across many different industries, a variety of flue gases will need to be tested.

In summary, research to date suggests mitigation of CO2 using algae cultivation is promising providing the gases are at a low concentration and contain low levels of contamination and operational conditions (e. g., pH, temperature, light) are controlled. Additionally the cost and energy input of transporting the gases to the ponds or bioreactors must be balanced by the benefits that the extra CO2 and the carbon mitigation provide. In a study investigating the potential for using power plant flue gas [54], it was estimated that an electricity consumption of 15.1 kWh/day was necessary for the direct injection of CO2 to one hectare of cultivation pond. The en­ergy benefits as a result of the injection would therefore need to be at least

15.1 kWh/d/ha and the CO2 savings greater than what would be emitted by conducting injection.

We have analysed the sustainable potential for the harvesting of woody biomass from forests for energy purposes, taking into consideration the demand for wood for other uses.

3.3.3.1 ADDITIONAL FOREST GROWTH

Sustainable additional forest growth is defined here as growth that is cur­rently not harvested and that:

• is not needed for future growth in demand for industrial roundwood, (e. g. for construction or paper production)

• can be harvested in an ecologically sound way.

The potential for sustainable additional forest growth was primarily based on a study by Smeets [2]. According to the study, the world’s techni­cal potential for additional forest growth would be ~64 EJ of woody bio­mass in 2050. However, the ecologically constrained potential is found to be ~8 EJ. The difference lies in the exclusion of all protected, inaccessible and undisturbed areas from the ecological potential. This means that only areas of forest classified as ‘disturbed and currently available for wood supply’ are included. A further sustainability safeguard is the use of only commercial species in the gross annual increment, rather than all available species.

Because the calculations by Smeets are partially based on an older source from 1998 [28], an additional calculation was done for a selection of six countries (Brazil, Russia, Latvia, Poland, Argentina and Canada). This was considered necessary because in some of these countries, the area of ‘disturbed’ forest could have considerably changed in the time pe­riod from 1998 until today.

In the additional calculation, the amount of sustainable complementary fellings resulting from the additional disturbed forest area available for wood supply, compared to the original base data, was determined, based on more recent country reports for the Global Forest Assessment 2010 (with data ranging from 2004 to 2008) [29]. This resulted in additional potential, particularly for Russia and Canada. The main differences were caused by the updated statistics on disturbed forest available for wood supply. The additional potential for the six countries was added to the eco­logical potential from [2], resulting in a total global potential for sustain­able additional forest growth of ~27 EJ.

Hirano et al. [26] observed acceleration of the rates of O2 evolution as well as synthesis of sugar during photosynthesis in Spirulina platensis when exposed to 10 mT geomagnetic field. They opined that the treatment using magnetic field increased the phycocyanin content in S. platensis, which plays an important role in the activation of photosystem II to help the ac­tivation of electron transfer reactions during photosynthesis. Their results also suggested that the magnetic fields accelerate the light excitation of chlorophyll radical pair.

Li et al. [27] subjected the same cyanobacterium S. platensis, to a range of static magnetic field intensities among which some stimulated its growth, uptake of carbon and light energy utilization. They observed that the levels of micro and trace elements (Ni, Sr, Cu, Mg, Fe, Mn, Ca, Co and V) and essential amino acids such as histidine improved at 250 mT magnetic field treatments. Also, chlorophyll a content of the magneti­cally treated sample was higher than the control, suggesting better light harvesting for photosynthesis. However there was slight decrease in lipid synthesis.

In Dunaliella salina, P-carotene content could be raised when treated with 10-23 mT of static magnetic field and the maximum was obtained at 10 mT with addition of 1 mg L-1 of Fe-EDTA. It also showed higher accumulation of the heavy metals viz. Co, Cd, Cu and Ni in the magneti­cally treated cultures, indicating its potential for bioremediation of heavy metals [32].

Singh et al. [29] investigated the use of permanent magnets and found that the physiological response of a cyanobacterium Anabaena doliolum, was dependent on exposure time and magnetic pole orientation. They reported that N, S and N+S poles from 0.3 T permanent magnets produced different ef­fects depending on the exposure time from 1 to 6 h. The effect was significant on a two hour exposure with combined N+S poles, where one culture was exposed to only N pole, which was then mixed with another culture exposed to S pole only. Treated cultures recorded 150, 110, 38, 34 and 20% increase in phycocyanin, chlorophyll a, carbohydrates, carotenoid and protein content, respectively and 55% increase in optical density over the control.

5.3.1.3 OTHER PHYSIOLOGICAL PROCESSES

Despite the potential advantages, more work is needed to optimize the resin approach. The use of resin beads is convenient for comparing prop­erties such as binding capacity and fouling for both commercial and our own laboratory-synthesized resins. Here we used a commercially avail­able strong cation exchange resin where sulfuric acid/methanol is used to elute the resin. It is entirely feasible to use a weakly basic anion exchange resin where raising the pH releases the algae from the resin [25]. At lower pH (7-8) these resins are positively charged and effectively bind algae. Raising the pH to ~pH 10 or above deprotonates the resin rendering it uncharged such that algae are released. Sodium methoxide in methanol, a strongly basic commercial transesterification reagent, would raise the pH, thereby releasing the algae and catalyzing the transesterification reaction. Here also, it would be critical to use a nonporous resin that did not entrain water.

Future directions are aimed at determining which approach, using strong or weak anion exchange resins is more effective in terms of binding capacity, fouling, and FAME yields. It will also be important to develop new modes for use of the resins such as thin film coatings or resin coated particles that do not entrain water. Finally, more studies of the transesteri­fication reaction are in order since, as shown with Neochloris, the one step conversion to FAME was not quite complete and was further affected by the nitrogen starvation.

As an alternative feedstock for biodiesel production, microalgae have the following advantages over conventional oil crops such as soybeans: (1) microalgae have simple structures, but high photosynthetic efficiency with a growth doubling time as short as 24 h. Moreover, microalgae can be produced all year round. Some data in Table 1 [4] show microalgae are the only source of biodiesel that have the potential to completely displace fos­sil diesel. (2) The species abundance and biodiversity of microalgae over a broad spectrum of climates and geographic regions make seasonal and geographical restrictions much less of a concern compared with other lipid feedstocks. Microalgae may be cultivated on freshwater, saltwater lakes with eutrophication, oceans, marginal lands, deserts, etc. (3) Microalgae can effectively remove nutrients such as nitrogen and phosphorus, and heavy metals from wastewaters. (4) Microalgae sequester a large amount of carbons via photosynthesis, for example, the CO2 fixation efficiency of Chlorella vulgaris was up to 260 mgL-1h-1 in a membrane photobio­reactor [5]. Utilization of CO2 from thermal power plants by large-scale microalgae production facilities can reduce a great deal of the greenhouse gas emissions blamed for global warming. (5) The production and use of microalgae biodiesel contribute near zero net CO2 and sulfur to the atmo­sphere. (6) Microalgae can produce a number of valuable products, such as proteins, polysaccharides, pigments, animal feeds, fertilizers, and so on. In short, microalgae are a largely untapped biomass resource for renew­able energy production.

However, commercialization of microalgae biomass and biofuel pro­duction is still facing significant obstacles due to high production costs and poor efficiency. In face of these challenges, researchers are undertak­ing profound efforts to improve microalgae biomass production and lipid accumulation and lower downstream processing costs.

Energy consumption in the production process is deemed the largest ob­stacle to algal biofuel production and a positive energy balance is a neces­sity but difficult to achieve. There are now several LCA studies which have investigated the amount of energy consumed in each of the neces­sary processes and comparing this to the energy recovery potential. Some studies have suggested that a positive energy balance is possible others suggest the contrary. Lardon et al. [77] found that if algae was cultivated purely for biodiesel, a positive balance would be unattainable. However if the residual biomass were to be anaerobically digested, a positive balance could be achieved in the scenario of growing algae in low nitrogen media and processing wet biomass [86].

The majority of other life-cycle analyses conducted recently suggest that algal biofuel can be produced with a positive energy balance though possibly not as positive as some alternative biofuels. Clarens et al. [87] modelled the growth of algae in raceway ponds and compared the energy consumption and environmental impacts of the fuel produced to fuel from corn, canola and switchgrass. In terms of energy consumption it was found that algal biodiesel required a far higher input, at least four times as much) as the next highest, and a sensitivity analysis revealed that the energy con­sumption was mainly a result of fertiliser use and carbon dioxide produc­tion [88]. Sander and Murthy [95] conducted a LCA comparing the differ­ence between harvesting methods of filter pressing and centrifugation, and a positive energy balance for both methods was reported, with a higher net energy yield for the filter press (almost double that of the centrifuge). The mentioned study did not provide details of the modelled strain nor likely productivity rates; it assumed that the algae contained 30% lipids, which would be difficult to achieve for an outdoor cultured strain; and did con­sider year round production, which would also be a challenge. In a study by Stephenson et al. [96] air-lift tubular photobioreactors and raceway ponds were compared in terms of energy consumption and yield, and their results suggested that the majority of energy was consumed in the cultivation stage (i. e., the cultivation stage in bio-reactors required approximately 10 times more energy than raceway ponds), which is in contrast to previous studies [77][87]. This study has similarities with that of Jorquera et al. [55] who showed raceway ponds provided a far greater energy balance than bioreactors. A high energy consumption in the bio-reactors was attributed to the manufacture of the PVC material and circulation of the culture [55]. The majority of energy consumed from cultivation in raceway ponds was due to circulation using a paddlewheel. It was suggested that for raceway cultivation the anaerobic digestion of the residual biomass could offset the energy required from cultivation, however this was not the case for the tubular photo-bioreactors [55].

In a further study conducted by Clarens et al. [97], different process chains and how these affect the energy balance or Energy Return on In­vestment (EROI) were compared, particularly the study looked at the vari­ous end-products from the algae (i. e., anaerobic digestion (AD) to electric­ity, biodiesel and AD to electricity, biodiesel and combustion to electricity and direct combustion) as well as source options for CO2 (i. e., virgin CO2, carbon capture, flue gas) and nutrients (wastewater supplementation). In each case direct combustion of the biomass to electricity produced the highest EROI and the best option was direct combustion of the biomass with direct compression of flue gas providing a source of CO2 [97]. The EROI for this scenario was 4.10, a similar scenario using flue gas and wastewater supplementation provided an EROI of 4.09. According to Cla­rens et al. [97] a value greater than 3 is considered sustainable, comparing well to canola (2.73), but not to switchgrass (15.90). Table 8 provides a summary of the best energy balances produced through the main studies conducted investigating energy recovery from algae.

Current production of biodiesel from algae without some other form of energy recovery will usually give a negative energy balance. To overcome this, it is necessary to include another form of energy recovery such as anaerobic digestion or combustion. It may even be far more beneficial to ignore biodiesel and to recover energy directly from anaerobic digestion or combustion as their input requirements are significantly lower. Energy reduction measures in each process will further improve the viability of biofuel from algae whatever the process stream used. Further work is re­quired to find the optimal recovery method that can compete with the en­ergy balance of conventional biofuels.

COLIN M. BEAL, ROBERT E. HEBNER, MICHAEL E. WEBBER, RODNEY S. RUOFF, A. FRANK SEIBERT, and CAREY W. KING

5.1 INTRODUCTION

The aspiration for producing algal biofuel is motivated by the desire to:

(1) displace conventional petroleum-based fuels, which are exhaustible,

(2) produce fuels domestically to reduce energy imports, and (3) reduce greenhouse gas emissions by cultivating algae that re-use carbon dioxide emitted from industrial facilities. In theory, algae have the potential to pro­duce a large amount of petroleum fuel substitutes, while avoiding the need for large amounts of fresh water and arable land [1-3]. These attributes have created widespread interest in algal biofuels. In practice, however, profitable algal biofuel production faces several important challenges. The goal of the research presented in this paper is to examine and quantify the extent of some of those challenges with an eye towards identifying critical areas for advances in the development of algal biofuels.

For algae to be a viable feedstock for fuel production: a significant quantity of fuel must be produced, the energy return on investment (EROI) of the life cycle must be greater than 1 (and practically greater than 3 [4]), the financial return on investment (FROI) should be greater than 1, the wa­ter intensity of transportation using algal biofuels should be sustainable, and nutrient requirements should be manageable. This study examines these criteria for two cases using second-order analysis methods described by Mulder and Hagens [5], which include direct and indirect operating

expenses, but neglect all capital expense. Process-specific terminology is based on the reporting framework established by Beal et al. [6].

There are several energy carriers and co-products that can be produced from algae, such as renewable diesel, electricity, hydrogen, ethanol, phar­maceutics, cosmetics, and fertilizers [7-9]. While non-energy co-products might enable economic viability of algal biofuel products in the short term, large scale production would quickly saturate co-product markets. Thus, in the long term, production of domestic, renewable, low-carbon fuels as an alternative to conventional fuel sources remains the main motivation for researching large-scale algae production. Consequently, this research focuses on the energy products. While bioelectricity from algal feedstocks is one possible pathway for energy production, this work considers only the co-production of bio-oil (a petroleum fuel substitute) and bio-gas (i. e., methane, which is a natural gas substitute) because those two fuels are produced from the experimental process at UT and align more directly with displacing petroleum [10-12]. Further, both bio-oil and bio-gas are feedstocks that can be combusted within additional technologies to pro­duce electricity.

Because the intent of this research is to analyze and anticipate a mature algal fuels industry that does not yet exist, researchers have two options for conducting a process analysis as in this paper: (1) use data derived from experimental processes followed by scaling analyses (recognizing that lab-scale experiments are inherently sub-optimal) or (2) use estimated data from models of future commercial-scale systems. Both of these ap­proaches are used in this study. Firstly, an Experimental Case is described, which is based on unique direct end-to-end measurements (from growth through biocrude separations) performed in a controlled indoor/outdoor laboratory setting at The University of Texas at Austin. Secondly, a High­ly Productive Case is described, which is based an optimistic analytical model that incorporates the technology and pathways of the Experimental Case.

We encourage other researchers to present (life cycle) metrics of al­ternative algal technology pathways in the step-by-step manner we dem­onstrate. The reasons for presenting life cycle metrics at multiple stages are threefold: (1) easier facilitation of future life cycle assessment (LCA) harmonization and meta-analyses that can effectively compare many independent studies, (2) better tracking of technological progress over time, and (3) better comparison of competing technologies (e. g., capital intensive versus resource intensive). The benefits of LCA harmonization were demonstrated by Farrell et al. [13] in comparing net energy for corn ethanol. The National Renewable Energy Laboratory of the US Depart­ment of Energy tests and tracks photovoltaic cell efficiencies over time such that specialists and the general public can easily track the rate of progress, which is beneficial for the community as a whole. By doing so, one is able to observe the improvements that were made to photovoltaic cell designs over the course of research and development, providing a van­tage point for researchers and investors alike to gauge the progress in that energy production technology. The authors believe algal energy processes would benefit from similar indicators and analyses, and this manuscript presents its results in that spirit of tracking technological metrics starting at the experimental batch scale. Additionally, the calculation of multiple life cycle indicators (e. g., EROI, FROI, water use, resource consumption, land use, air emissions, etc.) from the same experimental or modeled pro­cesses provides congruent indicators that emphasize the real design trad­eoffs (e. g., water versus electricity inputs).

The work presented adds to research in the authors’ prior publications, which presented the second-order energy return on investment (2nd O EROI) analyses for an Experimental Case and a modeled Highly Produc­tive Case. In the previous work, the 2nd O EROI, which is a ratio of the energy output of a system to the energy input for that system, for these two cases was determined to be 9.2 * 10-4 and 0.22, respectively [14]. That study illustrated the energetic challenges associated with producing algal biofuel. The present study extends the previous work with five new analytical thrusts to determine (1) the partial FROI, (2) the second-order water intensity of transportation using the algal biofuels produced, (3) the nitrogen constraints, (4) the carbon constraints, and (5) the electricity re­source constraints for the Experimental Case and the Highly Productive Case, respectively. The cost, water, and resource results from this new work are presented in conjunction with the previously determined energy results. Thus, for our two cases (one experimentally measured and one analytically derived), this present research serves as a comprehensive and coherent evaluation of the algal biofuel process. It is important that LCAs demonstrate relationships among multiple metrics that are calculated. By reporting multiple metrics for the same algal energy processes, this paper presents an understanding of how one metric (e. g., water consumption) is linked to another (e. g., energy production). Although the Experimental Case is not representative of commercial biofuel production due to signifi­cant artifacts that are inherent to lab-scale (vs. industrial scale) production, it represents the first known end-to-end experimental characterization of algal biocrude production at relatively large scale (thousands of liters). While other experiments have been performed at similar scale, they did not conduct the comprehensive mass and material balances that are pre­sented here. Conversely, the data used for the Highly Productive Case are based on optimistic assumptions for operating within the specific produc­tion pathway in this study. To place the Highly Productive Case in context with other analyses that have been published, each assumption is com­pared with those from other studies in the literature.

Many prior studies have been performed, each with a slightly different focus: some have emphasized algal biomass productivity, estimated algal oil productivity per acre of land, or evaluated only a few constraints on al­gal biofuel production (e. g., energy requirements, cost, etc.) [15-18]. This paper takes the approach of considering many constraints simultaneously (energy, cost, water, and resources) to give a more complete assessment. To this end, quantitative targets are presented in the “Conclusions” that, if achieved, would enable algal biofuel production at large scale.

These models explain biological processes considering electromagnetic fields as modulators of molecular information transfer. It is considered that the EMF either itself acts as signal(s) and/or intercepts or modifies the processes of molecular interaction.

6.4.2.1 ION CYCLOTRON RESONANCE CONCEPT

Many authors have developed the idea of ion cyclotron resonance (ICR) of specific ions like Ca2+ and Na+ [68] which predicts ELF magnetic effects at the cyclotron frequencies and there harmonics. Later, it was modified to the ion parametric resonance (IPR) model, which includes the cyclotron sub harmonics. The IPR is composed of a number of theoretical models based on classical and quantum electrodynamics where biomagnetic ef­fects are considered as magnetically modulated ion binding in ion-ligand interactions [69]. Free ions move with the cyclotron frequency in a static magnetic field and can be influenced by ELF magnetic fields or appropri­ate frequencies [70]. The main focus of these studies was the essential role of Ca2+ ions in magnetobiology experiments. It is proposed that ion behav­ior in channels like the acetylcholine receptor have constrictions in them, which cause thermal collisions. Under certain magnetic field parameters the wall collisions could be avoided at certain amplitudes and frequencies determined for the Lorentz force equation [71]. Under these conditions, the ions are predicted to “fly” through the channel unimpeded increasing the membrane permeability. ICR allows circulation of ions through selec­tive enhancement, which affects the rate of biochemical reactions [72].

The fact that magnetic fields can modulate enzyme activities in vitro is a crucial observation, because it indicates that enzymes may function as magnetoreceptors [69]. EMF modulations could also initiate changes in the distribution of protein and lipid domains in the membrane bilayer, as well as conformational changes in lipid-protein associations [1]. The interface between cell membrane and extra and intercellular fluids can be electrified on the order of 106 to 1010 V cm-1 [73]. The impact of an elec­tric field on a biological cell membrane and its change with time may constitute a relevant mechanism of information transmission influencing the membrane properties. The electric field, mainly generated by ions flowing to the membrane from the external environment, can change the molecular distribution of electronic charge inside each lipid molecule, producing perturbations of collective excitations in the mechanical and electrical properties of the lipid chain which can be treated as a mechanism for intermembrane communication, analogous to a damped harmonic os­cillations [74].

Pilla et al. [73] presented a working model of electrochemical informa­tion transfer by which the injection of low-level current can provide func­tional selectivity in the kinetic modulation of cell regulation. His theory was based on ion/ligand binding being a possible transduction mechanism for the detection of exogenous EMF’s at the cell membrane [75]. In or­der to derive the specifications for electromagnetic field signals having optimal biological effects, it is first necessary to develop a model for the underlying biological processes which are assumed to be complex physi­cal systems that may be modeled mathematically as non-linear, time-vary­ing, finite-dimensional dynamic systems. They developed a method for the systematic analysis of electrical impedance for each relevant electro­chemical pathway of a cellular system [62]. The electrochemical transfer hypothesis postulated that the cell membrane would be the site of interac­tion of low level electromagnetic fields by altering the rate of binding of calcium ions to enzymes or receptor sites [1]. The Ca2+ pathway can be influenced by EMFs on the complex chain of transduction, amplification, and expression.

Experimental results have shown that specific ion/ligand binding path­ways such as Ca2+ binding to calmodulin (CaM) and the ensuing steps of calcium-dependent signaling to intracellular enzymes may act as primary transduction mechanisms for EMF detection leading to an increase in the instantaneous reaction velocity and enzyme kinetics [75,76]. Calmodulin also plays a role in many other important biochemical processes such as cell proliferation, Ca2+ membrane transport and plant cell function [77]. An alteration of cell signaling events can lead to changes in cell prolifera­tion and differentiation, which can be initiated, promoted or co-promoted [70]. The capability of the weak EMF to have a bioeffect appears to reside in the informational content of the waveform [1]. The waveform duration and the voltage dependence are the most important parameters to increase the activity of the specific adsorption of an enzyme [62]. The proposed interfacial membrane model reveals that it is entirely reasonable to expect specific electrochemical effects as a result of electrical stimulation with signals of relatively low frequency and amplitude [73].

The incorporation of quantum states into ion interference has also been involved in the explanation of the physical nature of magnetoreception [78]. Variations in magnetic field magnitude affect the phase of ion wave functions and the interference of these phase changes affect the physi­cal observables in quantum mechanics. This theory predicts magnetobio­logical effects for magnitude/direction modulated magnetic fields, pulsed magnetic fields and weak AC electric fields among others [51]. In these cases, ions of calcium, magnesium, zinc, hydrogen, and potassium appear to be relevant. However, the most prominent example of a proven bio­electromagnetic mechanism is the radical pair recombination mechanism, which has been demonstrated biochemically in vitro. Radical pairs are formed as reaction intermediates in many biochemical reactions within complex reaction chains under the influence of exogenous electromag­netic influence [70]. The recent breakthrough regarding the radical pair mechanism in the blue light receptor protein, cryptochrome, by Schulten and his colleagues, supports the concept that radical pair recombination is involved in magnetoreception in avian navigation. Molecular model­ing and calculations showed that the signaling of cryptochrome, which involves a photoreduction process, can be modulated in the presence of a magnetic field on the order of 1 mT inducing an increase in the signal­ing activity of the protein by ~10% [79,80]. This prediction appears to be consistent with the experimental results on the effect of magnetic fields on cryptochrome-dependent responses in Arabidopsis thaliana seedlings attained by Ahmad and coworkers [81]. It is then suggested that the mag­netic navigation capability could be mediated by the presence of cryp­tochrome that is localized in the retinas of migratory birds which could alter how the bird perceives colors enabling something akin to an internal magnetic compass [82]. This radical pair mechanism is probably coupled with the alternative magnetite-based mechanism of magnetoreception and navigation, which poses that the Earth’s magnetic field exerts a minute mechanical force on the magnetite particles found in the upper beaks of migrating birds providing positional information due to fluctuations in the geomagnetic strength in different locations [83].