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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