INTRODUCTION

A few short decades ago, if someone told you that used cooking oil, in­edible plant materials, trash, algae, etc. would one day be crucial in job creation, improving global economies, keeping the air clean and reducing pollution, solving global energy needs, and in matters of national security, you could have easily classified such an individual as being out of touch with reality. Today, however, this is the new reality. This book describes how production and use of biofuels (defined as fuels produced from previ­ously living organisms) is helping meet this new reality. In particular, we look at biofuels from algae and aquatic plants.

The reader will explore how biomass, specifically sugars, nonedible plant materials, and algae (which are designated first, second and third generation biofuels respectively), are used in production of fuel. A de­scription of the feasibility of such projects, current methodologies, and how to optimize biofuel production is presented.

Ever since the oil crisis of the 1970s, tremendous efforts have been devoted into seeking alternative fuels for the modern industrial, transport, and agricultural systems as they are heavily dependent on oil. The world population continues to increase rapidly while emergent economies such as India and China coupled with the fast rate of urbanization have put a severe strain on the current sources of fuel. This has also led to a concomi­tant rise in pollution with far-reaching environmental impacts. It is this realization that has made it necessary to publish this book.

This book starts with a clear and succinct description of biofuel pro­duction from microalgae (also referred to as phytoplankton), the progress made in this field, limitations of current methodologies, and sustainability issues. The book then delves into the role of bioenergy in a fully sustain­able global energy system. In particular, it examines the supply potential and use of biomass with the aim of achieving a transition to a fully renew­able global energy system by 2050. Important factors such as land use, food security, residues, and waste are also addressed.

The text not only discusses common types of biofuels and relatively simple technologies involved, it goes into detail about advanced biofuel technologies in some very unique ways. It describes plausible ways of optimizing biofuel production and ends with a detailed and captivating look at future research involving gene discovery in biofuel production. This features technological advances that make it possible to economi­cally cultivate microalgae that have a high lipid or starch content. Other efforts devoted into optimizing specific microalgae strains and environ­ments in order to increase the per cell enrichment of lipids or starch are also discussed in vivid detail.

Chapter 1, by Wu and colleagues, explores the role of bioenergy in the global energy system. They argue that microalgae represent a sustainable energy source because of their high biomass productivity and ability to remove air and water born pollutants. This paper reviews the current status of production and conversion of microalgae, including the advantages of microalgae biodiesel, high density cultivation of microalgae, high-lipid content microalgae selection and metabolic control, and innovative har­vesting and processing technologies. The key barriers to commercial pro­duction of microalgae biodiesel and future perspective of the technologies are also discussed.

In chapter 2, Aitken and Antizar-Ladislao investigate the potential of producing biofuels from algae, which has been enjoying a recent revival due to heightened oil prices, uncertain fossil fuel sources, and legislative targets aimed at reducing our contribution to climate change. If the con­cept is to become a reality, however, many obstacles need to be overcome. It is necessary to minimize energetic inputs to the system and maximize energy recovery. The cultivation process can be one of the greatest en­ergy consumption hotspots in the whole system: recent studies suggest that open ponds provide the most sustainable means of cultivation infra­structure due to low energy requirements compared to more energy in­tensive photobioreactors. Much focus has also been placed on finding or developing strains of algae that are capable of yielding high oil concentra­tions combined with high productivity. Yet to cultivate such strains in open ponds is difficult because of microbial competition and limited radiation — use efficiency. To improve viability, the use of wastewater has been con­sidered by many researchers as a potential source of nutrients with the added benefit of tertiary water treatment; however productivity rates are affected and optimal conditions can be difficult to maintain year round. This paper investigates the process streams that are likely to provide the most viable methods of energy recovery from cultivating and processing algal biomass. The key findings are the importance of a flexible approach that depends upon location of the cultivation ponds and the industry tar­geted. Additionally this study recommends moving toward technologies producing higher energy recoveries such as pyrolysis or anaerobic diges­tion as opposed to other studies that have focused on biodiesel production.

Cornelissen and colleagues present a detailed analysis of the supply potential and use of biomass in the context of a transition to a fully renew­able global energy system by 2050 in chapter 3. They investigate bioener­gy potential within a framework of technological choices and sustainabil­ity criteria, including criteria on land use and food security, agricultural and processing inputs, complementary fellings, residues, and waste. This makes their approach more comprehensive, more stringent in the applied sustainability criteria, and more detailed on both the supply potential and the demand side use of biomass than that of most other studies. They find that the potential for sustainable bioenergy from residues and waste, com­plementary fellings, energy crops, and algae oil in 2050 is 340 EJ a-1 of primary energy. This potential is then compared to the demand for bio­mass-based energy in the demand scenario related to this study, the Ecofys Energy Scenario [1]. This scenario, after applying energy efficiency and non-bioenergy renewable options, requires a significant contribution of bioenergy to meet the remaining energy demand; 185 EJ a-1 of the 340 EJ a-1 potential supply. For land use for energy crops, they find that a maxi­mum of 2,500,000 km2 is needed of a 6,730,000 km2 sustainable potential. For greenhouse gas emissions from bioenergy, a 75%-85% reduction can be achieved compared to fossil references. They conclude that bioener­gy can meet residual demand in the Ecofys Energy Scenario sustainably with low associated greenhouse gas emissions. It thus contributes to its achievement of a 95% renewable energy system globally by 2050.

Chapter 4, by Alam and colleagues, argues that fossil fuel energy re­sources are depleting rapidly, and most importantly the liquid fossil fuel will be diminished by the middle of this century. In addition, the fossil fuel is directly related to air pollution and land and water degradation. In these circumstances, biofuel from renewable sources can be an alternative to re­duce our dependency on fossil fuel and assist to maintain the healthy glob­al environment and economic sustainability. Production of biofuel from food stock generally consumed by humans or animals can be problematic and the root cause of worldwide dissatisfaction. Biofuels production from microalgae can provide some distinctive advantages such as their rapid growth rate, greenhouse gas fixation ability, and high production capacity of lipids. This paper reviews the current status of biofuel from algae as a renewable source.

Worldwide, algal biofuel research and development efforts have fo­cused on increasing the competitiveness of algal biofuels by increasing the energy and financial return on investments, reducing water intensity and resource requirements, and increasing algal productivity. In chapter 5, Beal and colleagues present analyses in each of these areas—costs, re­source needs, and productivity—for two cases: (1) an experimental case, using mostly measured data for a lab-scale system, and (2) a theorized highly productive case that represents an optimized commercial-scale pro­duction system, albeit one that relies on full-price water, nutrients, and carbon dioxide. For both cases, the analysis described herein concludes that the energy and financial return on investments are less than 1, the water intensity is greater than that for conventional fuels, and the amounts of required resources at a meaningful scale of production amount to sig­nificant fractions of current consumption (e. g., nitrogen). The analysis and presentation of results highlight critical areas for advancement and inno­vation that must occur for sustainable and profitable algal biofuel produc­tion that can occur at a scale that yields significant petroleum displace­ment. To this end, targets for energy consumption, production cost, water consumption, and nutrient consumption are presented that would promote sustainable algal biofuel production. Furthermore, this work demonstrates a procedure and method by which subsequent advances in technology and biotechnology can be framed to track progress.

Hunt and colleagues explore the surge of interest in bioenergy in chap­ter 6. This interest has been marked with increasing efforts in research and development to identify new sources of biomass and to incorporate cutting-edge biotechnology to improve efficiency and increase yields. It is evident that various microorganisms will play an integral role in the development of this newly emerging industry, such as yeast for ethanol and Escherichia coli for fine chemical fermentation. However, it appears that microalgae have become the most promising prospect for biomass production due to their ability to grow fast, produce large quantities of lipids, carbohydrates and proteins, thrive in poor quality waters, sequester and recycle carbon dioxide from industrial flue gases, and remove pollut­ants from industrial, agricultural and municipal wastewaters. In an attempt to better understand and manipulate microorganisms for optimum produc­tion capacity, many researchers have investigated alternative methods for stimulating their growth and metabolic behavior. One such novel approach is the use of electromagnetic fields for the stimulation of growth and meta­bolic cascades and controlling biochemical pathways. An effort has been made in this review to consolidate the information on the current status of biostimulation research to enhance microbial growth and metabolism using electromagnetic fields. It summarizes information on the biostimula­tory effects on growth and other biological processes to obtain insight re­garding factors and dosages that lead to the stimulation and also what kind of processes have been reportedly affected. Diverse mechanistic theories and explanations for biological effects of electromagnetic fields on intra — and extracellular environment have been discussed. The foundations of biophysical interactions such as bioelectromagnetic and biophotonic com­munication and organization within living systems are expounded with special consideration for spatiotemporal aspects of electromagnetic topol­ogy, leading to the potential of multipolar electromagnetic systems. The future direction for the use of biostimulation using bioelectromagnetic, biophotonic and electrochemical methods have been proposed for biotech­nology industries in general with emphasis on an holistic biofuel system encompassing production of algal biomass, its processing, and conversion to biofuel.

Chapter 7 looks at how biomass can efficiently replace petroleum in the production of fuels for the transportation sector. Serrano-Ruiz and col­leagues argue that one effective strategy for the processing of complex biomass feedstocks involves previous conversion into simpler compounds (platform molecules) that are more easily transformed in subsequent up­grading reactions. Lactic acid and levulinic acid are two of these relevant biomass derivatives that can easily be derived from biomass sources by means of microbial and/or chemical routes. The present paper intends to cover the most relevant catalytic strategies designed today for the conver­sion of these molecules into advanced biofuels (e. g. higher alcohols, liquid hydrocarbon fuels) that are fully compatible with the existing hydrocar- bons-based transportation infrastructure. The routes described herein in­volve: (i) deoxygenation reactions that are required for controlling reac­tivity and for increasing energy density of highly functionalized lactic and levulinic acid combined with (ii) C C coupling reactions for increasing molecular weight of less-oxygenated reactive intermediates.

Jones and colleagues argue that some microalgae are particularly at­tractive as a renewable feedstock for biodiesel production due to their rapid growth, high content of triacylglycerols, and ability to be grown on non-arable land in chapter 8. Unfortunately, obtaining oil from algae is currently cost prohibitive in part due to the need to pump and process large volumes of dilute algal suspensions. In an effort to circumvent this problem, the authors have explored the use of anion exchange resins for simplifying the processing of algae to biofuel. Anion exchange resins can bind and accumulate the algal cells out of suspension to form a dewatered concentrate. Treatment of the resin-bound algae with sulfuric acid/metha — nol elutes the algae and regenerates the resin while converting algal lipids to biodiesel. Hydrophobic polymers can remove biodiesel from the sul­furic acid/methanol, allowing the transesterification reagent to be reused. They show that in situ transesterification of algal lipids can efficiently con­vert algal lipids to fatty acid methyl esters while allowing the resin and transesterification reagent to be recycled numerous times without loss of effectiveness.

Chapter 9 shows that biodiesel production from microalgae is being widely developed at different scales as a potential source of renewable energy with both economic and environmental benefits. Duong and col­leagues argue that although many microalgae species have been identi­fied and isolated for lipid production, there is currently no consensus as to which species provide the highest productivity. Different species are expected to function best at different aquatic, geographical, and climatic conditions. In addition, other value-added products are now being consid­ered for commercial production, which necessitates the selection of the most capable algae strains suitable for multiple-product algae biorefineries.

Here the authors present and review practical issues of several simple and robust methods for microalgae isolation and selection for traits that may be most relevant for commercial biodiesel production. A combination of conventional and modern techniques is likely to be the most efficient route from isolation to large-scale cultivation.

In chapter 10, Liu and colleagues show that with fast development and wide applications of next-generation sequencing (NGS) technologies, ge­nomic sequence information is within reach to aid the achievement of goals to decode life mysteries, make better crops, detect pathogens, and improve life qualities. NGS systems are typically represented by SOLiD/Ion Tor­rent PGM from Life Sciences, Genome Analyzer/HiSeq 2000/MiSeq from Illumina, and GS FLX Titanium/GS Junior from Roche. Beijing Genom­ics Institute (BGI), which possesses the world’s biggest sequencing capac­ity, has multiple NGS systems including 137 HiSeq 2000, 27 SOLiD, one Ion Torrent PGM, one MiSeq, and one 454 sequencer. The authors have accumulated extensive experience in sample handling, sequencing, and bioinformatics analysis. In this paper, technologies of these systems are reviewed, and first-hand data from extensive experience is summarized and analyzed to discuss the advantages and specifics associated with each sequencing system. At last, applications of NGS are summarized.

Lopez and colleagues explore progress in genome sequencing in chap­ter 11. This progress is proceeding at an exponential pace, and several new algal genomes are becoming available every year. One of the challenges facing the community is the association of protein sequences encoded in the genomes with biological function. While most genome assembly proj­ects generate annotations for predicted protein sequences, they are usually limited and integrate functional terms from a limited number of databases. Another challenge is the use of annotations to interpret large lists of “inter­esting” genes generated by genome-scale datasets. Previously, these gene lists had to be analyzed across several independent biological databases, often on a gene-by-gene basis. In contrast, several annotation databases, such as DAVID, integrate data from multiple functional databases and re­veal underlying biological themes of large gene lists. While several such databases have been constructed for animals, none is currently available for the study of algae. Due to renewed interest in algae as potential sources of biofuels and the emergence of multiple algal genome sequences, a significant need has arisen for such a database to process the growing compendiums of algal genomic data. The Algal Functional Annotation Tool is a web-based comprehensive analysis suite integrating annotation data from several pathway, ontology, and protein family databases. The current version provides annotation for the model alga Chlamydomonas reinhardtii, and in the future will include additional genomes. The site al­lows users to interpret large gene lists by identifying associated functional terms and their enrichment. Additionally, expression data for several ex­perimental conditions were compiled and analyzed to provide an expres­sion-based enrichment search. A tool to search for functionally related genes based on gene expression across these conditions is also provided. Other features include dynamic visualization of genes on KEGG pathway maps and batch gene identifier conversion. The Algal Functional Annota­tion Tool aims to provide an integrated data-mining environment for algal genomics by combining data from multiple annotation databases into a centralized tool. This site is designed to expedite the process of functional annotation and the interpretation of gene lists, such as those derived from high-throughput RNA-seq experiments. The tool is publicly available at http://pathways. mcdb. ucla. edu webcite.

In the final chapter, Mani-Yazdi and colleagues explore the lack of sequenced genomes for oleaginous microalgae; they argue that this lack limits our understanding of the mechanisms these organisms utilize to be­come enriched in triglycerides. Here, they report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous mi­croalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor. After growth under nitrogen replete and nitrogen limit­ing conditions, they quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant path­ways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite mea­surements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid syn­thesis pathway, TAG production during nitrogen limitation was bolstered by repression of the P-oxidation pathway, up-regulation of genes encod­ing for the pyruvate dehydrogenase complex that funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid bio­synthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. Their quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic engineering targets and strategies for focused efforts to improve the production rate and cellular content of biofuel pre­cursors in oleaginous microalgae.

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As mentioned above, open pond cultivation is currently considered the most viable option for large scale cultivation of algal biomass for energy. According to LCA research conducted by Jorquera et al. [55], open ponds provide a much higher net energy ratio in comparison to PBRs, both tu­bular and flat plated. Jorquera et al. [55] investigated the amount of en­ergy consumed and produced using the three different cultivation methods for a yield of 100,000 kg of biomass annually. Productivities and energy yields in PBRs were found about three times higher than ponds, due to differences in efficiency. The energy consumption of PBRs to produce the equivalent amount of algal biomass compared to cultivation in ponds was however considerably higher (ca. ten times for tubular PBRs). The high energy consumption of the PBRs is mainly due to the air pumping, water pumping and the caloric content of the equipment used. On the whole, it can be assumed that despite low productivity requirements, open ponds provide a higher biomass yield for the energy consumed. One limitation of this reported research [55] is that it assumed that the cultivation of algae and the estimated productivity in the pond is possible all year round.

It has been previously reported that, under environmental conditions, wild strains of algae are likely to dominate and the strain of algae will also change depending upon the season [91]. Before discussing controlled conditions further, it is important to stress that certain strains of algae may be controlled by their requirement for extreme conditions. Spirulina and Dunaliella, for example, require a high pH and raised salinity to survive, most invasive species would be intolerant of these conditions [91]. The majority of species, however, require less extreme conditions and compe­tition by native algae (and possibly other living microorganisms) remains a problem. During the summer seasons the dominant algae will be those that thrive in higher temperatures and conversely in winter those species that survive colder weather will dominate. Tseng et al. [92] found that at temperatures between 17 to 22 °C, Chlorella vulgaris dominated whereas at higher temperatures from 22 to 27 and even up to 32 °C, Scenedesmus ellipsoideus, S. dimorphus and Wastella botryoides were dominant. Al­ternative observations were, however, made by Professor Shelef finding that in Israel, Chlorella and Micratinium were dominant in the summer whereas Euglena and Scenedesmus were the common species in winter [23]. Clearly the location of cultivation has a large impact upon which species will dominate in each season. What is most important, however, is that when growing algae in environmental conditions, selectivity of strain is not currently possible.

Recently research has investigated methods of species control in cul­tivation ponds. For example, two high-rate algae ponds have been com­pared, one which included recycling of algae and one which did not [91]. The species under investigation was Pediastrum spp. and algal biomass was collected every day and settled in algal settling cones. One litre of biomass was returned to one of the ponds and not the other. The pond with recycling provided over 90% dominance of Pediastrum sp. in one year whilst the non-recycled pond provided only 53% dominance. It is sug­gested that this method of recycling may provide a useful method of spe­cies control in open pond cultivation, however it may not be successful for every strain required for cultivation. Further research is required to assess whether recycling will be beneficial for any considered strains.

The production of microalgae biomass for extraction of biofuels is gener­ally more expensive and technologically challenging than growing crops. Photosynthetic growth of microalgae requires light, CO2, water and in­organic salts. The temperature regime needs to be controlled strictly. For most microalgae growth, the temperature generally remains within 20°C to 30°C. In order to reduce the cost, the biodiesel production must rely on freely available sunlight, despite daily and seasonal variations in natural light levels [7, 17-20]. A number of ways the microalgae biomass can be converted into energy sources which includes: a) biochemical conversion, b) chemical reaction, c) direct combustion, and d) thermochemical con­version. Fig. 2 illustrates a schematic of biodiesel and bioethanol produc­tion processes using microalgae feedstock [10]. As mentioned previously, microalgae provide significant advantages over plants and seeds as they: i) synthesize and accumulate large quantities of neutral lipids (20 50 % dry weight of biomass) and grow at high rates; ii) are capable of all year round production, therefore, oil yield per area of microalgae cultures could greatly exceed the yield of best oilseed crops; iii) need less water than terrestrial crops therefore reducing the load on freshwater sources; iv) cul­tivation does not require herbicides or pesticides application; v) seques­ter CO2 from flue gases emitted from fossil fuel-fired power plants and other sources, thereby reducing emission of greenhouse gas (1 kg of dry algal biomass utilise about 1.83 kg of CO2). In addition, microalgae offer wastewater bioremediation by removing of NH4, NO3, PO4 from wastewa­ter sources (e. g. agricultural run-off, concentrated animal feed operations, and industrial and municipal wastewaters). Their ability to grow under harsher conditions and reduced needs for nutrients, microalgae can be cul­tivated in saline/brackish water/coastal seawater on non-arable land, and do not compete for resources with conventional agriculture. Depending on the microalgae species other compounds may also be extracted, with valuable applications in different industrial sectors, including a large range of fine chemicals and bulk products, such as polyunsaturated fatty acids, natural dyes, polysaccharides, pigments, antioxidants, high-value bioac­tive compounds, and proteins [2, 8, 10, 21-28].

Microalgal Biomass

FIGURE 2: Biofuel production processes from microalgae biomass, adapted from [2, 11]

i

Nutrients C02

FIGURE 3: Biodiesel and Bioethanol production processes from microalgae, adapted from [2]

There are different ways microalgae can be cultivated. However, two widely used cultivation systems are the open air system and photobiore­actor system. The photoreactor system can be sub-classified as a) tabular photoreactor, b) flat photoreactor, and c) column photoreactor. Each sys­tem has relative advantages and disadvantages. More details about these cultivation systems can be found in [2-3, 7].

The production of biofuel is a complex process. A schematic of biofuel production processes from microalgae is shown in Figure 3. The process consists of following stages: a) stage 1 — microalgae cultivation, b) stage 2 harvesting, drying & cell disruption (cells separation from the growth me­dium), c) stage 3 — lipid extraction for biodiesel production through trans­esterification and d) stage 4 starch hydrolysis, fermentation & distillation for bioethanol production. However, these processes are complex, techno­logically challenges and economically expensive. A significant challenge lies ahead for devising a viable biofuel production process [2, 28-30].

Above observations show growth stimulation by magnetic treatment in a diverse array of organisms (from prokaryotic to eukaryotic) and a va­riety of stimulative responses by each organism under varied conditions of treatment and growth. While former indicates at some general mode of mechanism(s), the later gives an impression in contrast to it. Lack of adequate information eludes a consensus on the mechanism(s). Several factors appear to be affecting the stimulation process. The flux generat­ing system, intensity of the flux, type of the flux (oscillatory or static), orientation of magnetic poles, duration of exposure, cell density and cell environment (for example type of medium and its ingredients) and other physicochemical conditions affect the process of biostimulation through electromagnetic forces. It has also been marked that the results sometimes do not show repeatability at other locations suggesting that local geomag­netic realities might also affect the process of stimulation.

FIGURE 4: Concept map of an EMF bio stimulation at different levels of living systems.

There are physiological effects other than growth that have been ob­served. These are processes such as carbon uptake, sugar synthesis and oxygen evolution in photosynthesis, synthesis of pigments (chlorophyll, carotenoids and phycocyanins), carbohydrates and proteins, accumulation of micro and trace metals and essential amino acids, fermentative activ­ity and even genetic processes like transposition. They can be stimulated under specific conditions adopted in the experiments. Only one study [29] specifically referred to lipids reported a decline in lipid content under the particular set of treatment. It may be worth noting that an exposure to surprisingly low levels of exogenous electromagnetic fields can have a profound effect on a large variety of biological systems [1]. A number of mechanisms have been proposed for observable magnetobiological and bioelectromagnetic effects at different levels [51]. A concept map, demon­strating different levels of the EMF influence is shown in Figure 4

VAN THANG DUONG, YAN LI, EKATERINA NOWAK, and PEER M. SCHENK

9.1 INTRODUCTION

Microalgae have been considered for biodiesel production, based on their ability to grow rapidly and to accumulate large amounts of storage lipids, primarily in the form of triacylglycerides (TAG). Microalgae are a group of mostly photoautotrophic microorganisms that includes both prokaryotic and eukaryotic species. These organisms can photosynthetically convert CO2 and minerals to biomass, but some species also grow heterotrophi — cally. Prokaryotic microalgae are cyanobacteria (blue-green algae) and eukaryotic microalgae include the nine phyla Glaucophyta, Chlorophyta, Chlorarachniophyta, Euglenophyta, Rhodophyta, Cryptophyta, Hetero — kontophyta, Haptophyta and Dinophyta. To date, about 2/3 of 50,000 species have been identified and are kept in collection by various algal research institutes [1]. For example, the largest collection at present is the Collection of Freshwater Algae at the University of Coimbra, Portu­gal, maintaining about 4000 strains and 1000 species of algae; the Cul­ture Collection of Algae of the Gottingen University, Germany harbors 2213 strains and 1273 species of both freshwater and marine algae; the

Culture Collection of Algae in the University of Texas, USA, maintains 2300 strains of freshwater species; the National Institute for Environmen­tal Studies in Japan is keeping 2150 strains with about 700 species of freshwater and marine algae [2]; the Australian National Algae Culture Collection (ANACC) maintains about 1000 strains of microalgae which were mostly isolated from Australian waters [3]. Although algae collec­tions are maintained for many purposes (e. g., for pharmaceutical, food, energy and industrial products), only a few hundred strains have been in­vestigated for chemical content and very few are cultivated in industrial quantities. To date, although there is mounting interest to develop micro­algal biodiesel production, the cost for microalgal biomass production is currently much higher than from other energy crops [4]. Thus, selection of an energy and cost-efficient production model could play a very important role in achieving competitive biodiesel production. This includes the se­lection of high lipid-producing algae, suitable farming locations, efficient cultivation and harvesting methods and oil extraction procedures. Here, we focus on the first step, the selection of suitable high lipid-accumulating microalgae strains, a process that can be compared with the early domes­tication of current crop plants. In alignment with this purpose, this review aims to present a practical guide to several simple and robust methods for microalgae isolation and selection for traits that maybe most relevant for commercial biodiesel production.

Microalgae cells are small (typically in the range of Ф2-70 pm) and the cell densities in culture broth are low (usually in the range of 0.3-5 gL-1). Harvesting microalgae from the culture broth and dewatering them are en­ergy intensive and therefore a major obstacle to commercial scale produc­tion and processing of microalgae. Many harvesting technologies, such as centrifugation, flocculation, filtration, gravity sedimentation, floatation, and electrophoresis techniques have been tested [45]. The choice of har­vesting technique depends on, in part, the characteristics of microalgae (such as their size and density) and the target products.

Following biomass harvesting it is then necessary to extract the maximum energy possible from the biomass to provide the best return. The three main fuel types that have received the majority of research related to algal biomass are biodiesel, biogas and bioethanol. Due to the potentially high oil content of certain strains of algae, the ease of extraction and value of the end product biodiesel has received the most attention. Algal strains such as Chlorella are noted for being able to produce up to 70% oil content within their cell walls [14]. This scenario however requires very specific conditions (low nitrogen and no contamination) and would be very diffi­cult to obtain in practice. It has been suggested by Sialve et al. [85] that it would not be economically viable to extract lipids from algae containing an oil yield any less than 40%, and therefore for the majority of algal spe­cies anaerobic digestion would provide the highest positive energy bal­ance due to low input requirements. Similarly in their environmental study Clarens et al. [97] found that direct combustion of biomass to produce electricity provided the highest energy return on investment when com­pared to anaerobic digestion, biodiesel production plus anaerobic diges­tion and biodiesel production plus direct combustion.

Given the high energy consumption required to produce biodiesel from algae, the process does not seem beneficial to be used for a flexible system where the algae cultivated are likely to be a mix of species with low lipid content. Bioethanol has potential and in such a system the algae is likely to contain a high proportion of convertible carbohydrates but the energy balance of such a process is untested and is unlikely to yield great efficien­cies in the near future and is likely to require high inputs (enzymes and yeast). It seems far more likely that anaerobic digestion or combustion of the biomass will provide the maximum energy recovery. Another benefit of such a concept is that facilities to carry out the digestion or combus­tion are likely to be already operating on site with no requirement for new development.

The 2nd O EROI for the Experimental Case and the Highly Productive Case, which have been reported previously by Beal et al. [14], are 9.2 * 10-4 ± 3.3 * 10-4 (cf. [19] for uncertainty analysis) and 0.22, respec­tively.

For algal biofuels to be produced commercially, the EROI must be competitive with that of conventional fuels (e. g., over the last few decades the EROI for oil and gas, including industrial capital, has typically been 10-20 [36] with delivered gasoline between 5 and 10 [37]). Several other studies have presented hypothetical energy analyses of algal biofuel pro­duction, and although the scope and systems evaluated vary, each of these studies has also found that without discounted inputs, the EROI is not competitive with conventional fuels [11,15,17,27,38]. The 2nd O EROI results from this study are plotted in Figure 2, along with the first-order EROI, which only includes direct energy inputs (and thereby neglects en­ergy embedded in material inputs).

For the Experimental Case, 90% (2308 kJ/Lp) of the total energy in­put (2572 kJ/Lp) was associated with bioreactor lighting, air compression (for supplying CO2), and pond mixing; all of which are considered to be artifacts of inefficient research-scale growth methods. Conversely, in the Highly Productive Case, which modeled efficient growth equipment, em­bedded energy in nutrients accounted for 85% (63 kJ/Lp) of the total energy
input (75 kJ/Lp). The Highly Productive Case assumes 8 kg of CO2, 70 g of nitrogen, and 8 g of phosphorus consumed per kg of algae produced.

Based on conservation of mass, the minimum possible CO2, nitrogen, and phosphorus consumption can be approximated as 1.8 kg, 70 g, and 8 g per kg of generic algal biomass, respectively [2,15,17,44-46]. Using these minimum data, and the associated energy equivalents (with values of 7.3 MJ/kg CO2, 59 MJ/kg N, and 44 MJ/kg P [15,19,46-51]), the mini­mum possible energy embedded in the (full-price) nutrients alone requires more energy (17.7 kJ/Lp) than the total energy produced (16.6 kJ/Lp), which prevents a positive net energy yield, and illustrates the need to use waste forms of nutrients. The energy embedded in carbon, nitrogen, and phosphorus is dependent on the stoichiometric requirement and energy intensity of production for each element. However, the embedded energy in these elements is independent of growth rate [14], demonstrating the limited ability for growth optimization to alter the overall EROI for algal biofuels.

The EROI was adjusted using quality factors reported by Beal et al. [14] that were calculated according to the price of each input, yielding a QA 2nd O EROI that directly parallels the PFROI analysis. For the Experi­mental Case and the Highly Productive Case, the QA 2nd O EROI was 9.2 x 10-5 and 0.36, respectively [14].

Water is well known to be an anomalous substance and plays a great role in living organisms. Due to the critical role water plays in biochemical and biological reactions, many studies have focused on the effects of magnetic and electromagnetic fields on water molecules [51]. These experiments have shown that water previously exposed to electrical, magnetic, electromag­netic, acoustic or vibrating fields keeps the acquired biological activity for extended periods of time [151]. Liquid water is clearly a very complex system when considering the complexity of molecular clusters, gas-liquid and solidliquid surfaces, reactions between the materials and the conse­quences of physical and electromagnetic processing [152].

The investigation of indirect magnetic field effects have shown that magnetically treated water has changes in light absorption, specific electri­cal conductivity, magnetic susceptibility, Raman spectrum, index of light refraction, surface tension and viscosity. The exposure of water to a static magnetic field is connected with the energy influence of the field on the water and biostructures. Markov [153] has also shown that static magnetic fields influence the speed of protoplasm movement, the miotic activity, and the quantity of pigments such as chlorophyll a, b and organic acids in plants. Water stores and transmits information concerning solutes, by means of its hydrogen-bonded network. The conditioning of water via per­manent magnetic and electromagnetic oscillating fields has been found to be stimulatory or inhibitory depending on the residence time of the wa­ter. S. cerevisiae exhibited the strongest influence by measuring a growth rate increase of ~60% after exposing the culture media to 15-30 seconds of a 100 kHz EMF at 2 pT. Longer exposure times that were inhibitory, could become stimulatory after dilution suggesting the existence of active agent(s) generated by the field exposure. Increases in toxicity after apply­ing a biocide compared to a biocide+EMF indicates an enhanced cell wall permeability [154].

Ultra high dilutions are special preparations of a specific compound dissolved in a medium (usually water) that undergo dramatic dilutions (usually thirty 1:100 dilutions) that exceed Avogadro’s number such that the final dilution is void of any original dissolved molecules. Each dilution step is accompanied by some activation force, usually mechanical succus — sion (shock wave) or vigorous mixing. However, other experiments have used sonication, high-voltage electromagnetic pulses, passive or active resonant circuits. The experimental results indicate that “pure” water sam­ples can retain specific information regarding a “donor” substance which can be quantitatively measured via thermoluminescence, delayed lumi­nescence, excess heat-of-mixing/microcalorimetry, changes in pH and conductivity, alterations to FTIR spectra, enzymatic activity, and modula­tion of chemical, biochemical, and biological processes usually in accord with the donor substance. These experiments have been carried out with biological bioassays with dinoflagellates comparing succussed media, and modulation to Ca2+ channel affinity by non-thermal microwave exposure, as well as investigating physico-chemical effects on purely chemical sys­tems using ultra-high dilution of lithium chloride, sodium chloride, mer­curic chloride, and mercuric iodide [155-168].

It has been proposed that the water molecules respond to incident EMF exposure and form metas water states [164]. The experiments with ther­moluminescence, microcalorimetry, and conductivity measurements indi­cate molecular cluster formation, most likely originating from the hydro­gen bond network. The evolution of these physico-chemical parameters with time suggests a trigger effect on the formation of molecular aggre­gates following the potentization procedure [159]. The various initial per­turbations initiate development of a set of chain reactions of active oxygen species in water. Energy in the form of high-grade electronic excitations is released in reactions, which can support non-equilibrium state of an aqueous system [169]. Within these solutions, the molecular aggregates or clusters consisting of water molecules are connected by hydrogen bonds, in far from equilibrium conditions, which can remain in, or move away from their uns equilibrium state dissipating energy from the external en­vironment in the manner Prigogine has described “dissipative structures” [170]. The lifetime of a particular cluster, containing specific water mol­ecules will be not much longer than the life of individual hydrogen bonds, i. e., nanoseconds, but clusters can continue forever although with constant changing of their constituent water molecules [152]. However, the pri­macy of hydrogen bonds for the molecular aggregate structures is not es­sential, as the formation of H-bonded molecules are considered coherence domains in water by Coherent Quantum Electrodynamic Theory, where the H-bond dynamics are transferred to the origin of their pair potentials interacting with zero-point fluctuations of the A-field [171]. The existence of these physicochemical and biological effects from water should elevate water from its traditional role as a passive space-filling solvent in organ­isms, to a position of singular importance, the full significance of which is yet to be fully elucidated [143].

All autotrophic algal species require a source of nutrients. The most im­portant nutrients, (i. e., those that are needed in greatest concentrations) are nitrogen and phosphorous, but many other nutrients and trace metals are also necessary for optimal growth [16]. There are many media recipes designed to provide complete nutrition for most species of algae. Nutri­ent rich effluents however are often capable of providing almost all of the nutrients required by certain algal species and [17, 18] consequent culti­vation in effluent provides two significant benefits. Firstly, direct uptake of these nutrients and metals, produces cleaner water. Secondly, the algae generate oxygen which aids aerobic bacterial growth leading to additional metal and nutrient assimilation. In the 1950s experiments were carried out by Oswald and his colleagues investigating the symbiotic relationship be­tween algae and bacteria for wastewater treatment in oxidation ditches [3, 19-22]. The experiments which were undertaken used the algae Euglena sp. due to their natural presence in ditches under examination. Oswald and his colleagues discovered that the bacteria and algae in the oxidation ditch develop a symbiotic relationship producing a more stable and less hazard­ous effluent [21]. The economic potential of the algal cells for livestock feed was identified, and the merits of using a faster growing species of algae in effluent specifically for the purpose of livestock feed were dis­cussed [19]. Particular attention in the late 1950s was given to the design of wastewater treatment ponds and relationships between oxygen produc­tion, biological oxygen demand (BOD) removal and light use efficiency over specific periods of time as a function to a variety of species, depths of pond, treatment time, loading rates to identify optimal operational condi­tions [3]. The importance of algae in a heavily loaded oxidation pond to provide the necessary oxygen for sludge oxidation was highlighted, and this early research remains of particular interest as it identified optimal conditions for effluent treatment and demonstrated that oxidation ponds using the symbiotic relationship can achieve considerable BOD removal (>85%) [3].

Further important work was carried out on the use of algae in waste­water treatment in the 1970s and 1980s. Of particular interest was the research lead by Professor Shelef, with a focus on the growth of domi­nant species of algae in open ponds using raw sewage as the main source of nutrients [23]. His research indicated that Micractinium and Chlorella dominated in most cases, with retention times of around three to six days. Using an influent with concentrations of total suspended solids (TSS) ca. 340 mg/L and BOD ca. 310 mg/L, a considerable reduction to levels ca. 60 mg/L TSS and below 20mg/L BOD was reported. Additionally phos­phate was reduced to very low levels and around 10-40 mg/L ammonia remained. This resulted in low levels of organic contamination which al­lowed the use of the treated wastewater for irrigation, and the residual nutrients provided a source of fertiliser as an added value.

In the 1980’s the concept of growing algae was further developed how­ever the focus turned to utilising the biomass for fuel production due to the energy crisis in the United States at the time. Oswald continued his re­search and was joined by Dr. Benemann and together they investigated the potential for the cultivation of algae on a large scale for fuel production. During this time most research moved away from wastewater treatment and more towards high productivity of biomass and high fuel yields with work conducted on the use of flue gas as a source of carbon dioxide to in­crease productivity and provide a method of carbon mitigation. Due to the continued search however for a more economically and environmentally viable system, once again research has turned to the potential benefits of growth in wastewater. Several different wastewater types have been inves­tigated, most common are domestic sewage [24-27], agricultural wastewa­ter (swine and cattle) [26, 28-31] and several industrial wastewaters, e. g., carpet manufacturing [32] and distillation [33]. Previous research suggests that cultivation in the tertiary treatment stages of wastewater treatment may provide the ideal conditions for reasonable algal growth due to high residual nutrient loading and prior removal of organic contaminants [25]. In such a scenario algae can provide an effective means of nutrient polish­ing. Various strains of algae have been shown to effectively remove nitro­gen and phosphorous forms in a number of synthetic and actual wastewa­ters (Table 1).

The table displayed in Table 1 suggests that there is considerable po­tential for nutrient removal in wastewaters with high nutrient loading us­ing a variety of species of common algae and mixtures of locally dominant algae. Much of the research reported has however been conducted at a lab scale in photo-bioreactors which provide controlled conditions (e. g., tem­perature, light and species control) and therefore will improve the produc­tivity of the algae and thus the uptake rate of nutrients. There are however exceptions where polycultures of dominant algae have been grown both using an outdoor turf scrubber [34] and high rate algal ponds [35] as the culture methods. In the cases where biomass has been cultivated on turf scrubbers or in ponds the system has developed using species which come to dominate as species selection is not possible. Despite the lack of con­trollability of species dominance and environmental factors, these studies still demonstrate good N and P removal alongside reasonable productivity rates of biomass.

With many industries looking for cost effective means of nutrient re­moval from wastewaters, the cultivation of algal biomass can provide a suitable method which may return a positive balance of energy as opposed to conventional processes which generally require an energetic input.