Tai ngay!!! Ban co the xoa dong chu nay!!! 16990153267121000000 Algae for Biofuels and Energy Developments in Applied Phycology Series Editor: Michael A Borowitzka School of Biological Sciences and Biotechnology Murdoch University, Murdoch, Western Australia, Australia For further volumes: http://www.springer.com/series/7591 Michael A Borowitzka • Navid R Moheimani Editors Algae for Biofuels and Energy Editors Michael A Borowitzka Algae R&D Centre School of Biological Sciences and Biotechnology Murdoch University Murdoch, WA, Australia Navid R Moheimani Algae R&D Centre School of Biological Sciences and Biotechnology Murdoch University Murdoch, WA, Australia ISBN 978-94-007-5478-2 ISBN 978-94-007-5479-9 (eBook) DOI 10.1007/978-94-007-5479-9 Springer Dordrecht Heidelberg New York London Library of Congress Control Number: 2012954780 © Springer Science+Business Media Dordrecht 2013 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein Cover design: Dr Roberto de Philippis Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Preface The concept of using algae as a source of renewable fuels and energy is quite an old one, dating back at least to 1931, but one which gained much attention during the 1990’s oil crisis and then, once again, more recently interest in algae as a source of biofuels has risen dramatically The potential attractive features of algae have often been listed, but as yet the high cost of producing algae biomass means that algal biofuels as an economical, renewable and sustainable source of biofuels and bioenergy is still somewhat off in the future Microalgae are currently probably the most studied potential source of biofuels, and in the US alone there are some 30+ companies working in the area and total investment in R&D is in excess of several billion $US worldwide This book focuses on microalgae rather than seaweeds, as microalgae are the most attractive for renewable energy production, especially the production of biodiesel, although seaweed biomass can also be used The aim of this book is to review in detail the most important aspects of the microalgae-to-bioenergy process, with an emphasis on microalgae as sources of lipids for the production of biodiesel and as potential sources of hydrogen The book is meant as a guide and resource for both the experienced practitioners in the field and to those newer to this exciting field of research However, no single book can cover all aspects of the production of bioenergy from algae; for example, we not cover the fermentation of algal biomass to produce methane, nor the fermentation of algal sugars to ethanol or butanol This book begins (Chap 1) with an introduction to the history and developments over the last 80 years or so in the area of large-scale and commercial-scale culture of microalgae and the extensive literature that is available Much can be learned from the extensive research that has been carried out, and by knowing this history (some of which is not easily accessible) we can avoid repeating past mistakes One of the key attractions of microalgae is the high lipid content of some species and the lipid and fatty acid composition and metabolism is covered in Chap by Guschina and Harwood, and the production and properties of biodiesel from these algal oils is considered in detail by Knothe in Chap 12, while Chap by Peters et al considers hydrogenases, nitroginases and H2 production by water-oxidizing phototrophs (i.e algae and cyanobacteria) The first step in developing an algae bioenergy process is species and strain selection and this topic is considered in detail in Chap Chapter by Beardall and Raven focuses on light and inorganic carbon supply as key limiting factors to growth in dense cultures and Chap by Rasala et al looks at how genetic engineering may be used to improve and modify algae strains The systems for production of microalgae biomass are reviewed in Chaps (photobioreactors; Chini Zittelli et al.), (open pond systems (Borowitzka & Moheimani) and (systems utilizing waste waters; Craggs et al.) The key downstream processes of harvesting and dewatering and extraction of the lipids are covered in Chaps 10 (Pahl et al.) and 11 (Molina Grima et al.) Finally, Chap 13 (Jacobi and Posten) looks at the energy balances of closed photobioreactors and how these may be improved, Chap 14 (Flesch et al.) looks at the greenhouse gas balance of algae based biodiesel using a range of models, and Chap 15 (Borowitzka) describes the process of techno-economic modelling and how it can be used to guide R&D in the development of algae biofuels v vi Preface In our experience there is also often some confusion on the basic laboratory methods used in algae culture and for the analysis of their basic composition, and we have therefore included a chapter on these basic methods as used and verified in our laboratory over many years We hope that, by providing this information in an easily accessible format, newer workers in the field will be able to produce more reliable results which can then be easily compared between different laboratories Michael Armin Borowitzka Navid Reza Moheimani Contents Energy from Microalgae: A Short History Michael A Borowitzka Algal Lipids and Their Metabolism Irina A Guschina and John L Harwood 17 Hydrogenases, Nitrogenases, Anoxia, and H2 Production in Water-Oxidizing Phototrophs John W Peters, Eric S Boyd, Sarah D’Adamo, David W Mulder, Jesse Therien, and Matthew C Posewitz 37 Species and Strain Selection Michael A Borowitzka 77 Limits to Phototrophic Growth in Dense Culture: CO2 Supply and Light John Beardall and John A Raven 91 Genetic Engineering to Improve Algal Biofuels Production Beth A Rasala, Javier A Gimpel, Miller Tran, Mike J Hannon, Shigeki Joseph Miyake-Stoner, Elizabeth A Specht, and Stephen P Mayfield 99 Photobioreactors for Microalgal Biofuel Production 115 Graziella Chini Zittelli, Liliana Rodolfi, Niccoló Bassi, Natascia Biondi, and Mario R Tredici Open Pond Culture Systems 133 Michael A Borowitzka and Navid Reza Moheimani Wastewater Treatment and Algal Biofuel Production 153 Rupert J Craggs, Tryg J Lundquist, and John R Benemann 10 Harvesting, Thickening and Dewatering Microalgae Biomass 165 Stephen L Pahl, Andrew K Lee, Theo Kalaitzidis, Peter J Ashman, Suraj Sathe, and David M Lewis 11 Solvent Extraction for Microalgae Lipids 187 Emilio Molina Grima, María Jose Ibáñez González, and Antonio Giménez Giménez 12 Production and Properties of Biodiesel from Algal Oils 207 Gerhard Knothe 13 Energy Considerations of Photobioreactors 223 Anna Jacobi and Clemens Posten 14 Greenhouse Gas Balance and Algae-Based Biodiesel 233 Anne Flesch, Tom Beer, Peter K Campbell, David Batten, and Tim Grant vii viii Contents 15 Techno-Economic Modeling for Biofuels from Microalgae 255 Michael A Borowitzka 16 Standard Methods for Measuring Growth of Algae and Their Composition 265 Navid Reza Moheimani, Michael A Borowitzka, Andreas Isdepsky, and Sophie Fon Sing Index 285 Contributors Peter J Ashman School of Chemical Engineering, The University of Adelaide, Adelaide, SA, Australia David Batten Low Cost Algal Fuels, CSIRO Energy Transformed Flagship, Aspendale, VIC, Australia Niccoló Bassi Fotosintetica & Microbiologia S.r.l., Firenze, Italy John Beardall School of Biological Sciences, Monash University, Clayton, VIC, Australia Tom Beer Transport Biofuels Stream, CSIRO Energy Transformed Flagship, Aspendale, VIC, Australia John R Benemann Benemann and Associates, Walnut Creek, CA, USA Natascia Biondi Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Firenze, Italy Michael A Borowitzka Algae R&D Centre, School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA, Australia Eric S Boyd Department of Chemistry and Biochemistry, and Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, MT, USA Peter K Campbell University of Tasmania, Hobart, TAS, Australia Rupert J Craggs National Institute for Water and Atmospheric Research, Hamilton, New Zealand Sarah D’Adamo Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO, USA Anne Flesch Veolia Environnement, Paris, France Sophie Fon Sing Algae R&D Centre, School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA, Australia Antonio Giménez Giménez Department of Chemical Engineering, University of Almería, Almería, Spain Javier A Gimpel Division of Biological Sciences, University of California San Diego, San Diego, CA, USA Tim Grant Life Cycle Strategies, Melbourne, VIC, Australia Emilio Molina Grima Department of Chemical Engineering, University of Almería, Almería, Spain Irina A Guschina School of Biosciences, Cardiff University, Cardiff, Wales, UK ix 274 N.R Moheimani et al Method 16.2 Dry weight and ash-free (total organic) dry weight determination GF/C filter pretreatment For dry weight and ash-free dry weight determination the glass fibre filters used to concentrate the algae must be pre-combusted Pre-combust Whatman GF/C (2.5cm diameter) filters at 100°C for h Store filters in vacuum dessicator over KMnO4 crystals until use Precautions The dried algae can be very hygroscopic and care must be taken during weighing If the weight seems to be increasing slightly during weighing then re-dry the sample in the dessicator for 24 h over fresh KOH or another dessicant DRY WEIGHT DETERMINATION Carefully weigh pre-combusted filters to decimal places Place filters in filter unit and filter culture (generally about 10 mL of culture) until filter appears completely dry Wash filter with 10 mL of distilled water (freshwater spp) or isotonic ammonium formate solution for saline species (0.65 M for marine spp) Remove filter from filter unit and dry at 100°C for h and then place in vacuum desiccator over dessicant overnight Weigh dried filter containing algae to decimal places using a digit balance Dry weight (DW) = (weight of filter plus algae) - (weight of filter) ORGANIC DRY WEIGHT (ASH-FREE DRY WEIGHT) DETERMINATION Take filters from step of dry weight determination and ash at 450°C in a muffle furnace for h Cool filters in a vacuum dessicator over dessicant Rapidly and carefully weigh filter Ash-free dry weight (AFDW) = Dry Weight - Weight after ashing 16 Standard Methods for Measuring Growth of Algae and Their Composition 275 Method 16.3 Sample preparation steps for analysis of proximate composition Precautions : Some care must be taken when filtering delicate cells such as those of Dunaliella so that they are not ruptured during filtration leading to a loss of cell components Filter known amount of culture on GF/C or GF/F filters (In the case of dry weight measurement of marine and halophilic algae, precombusted filters must be used [see Method 16.2] and after filtration of the algae the filter must be rinsed with 10 mL of isotonic ammonium formate or 10mL acidic (pH 5.5) ammonium formate) Fold filter (algae side inwards) + dry with paper towel + freeze at -20°C (Freezing helps extraction but is not required for dry weight determination unless the filters are to be stored before analysis) If no cell wall (i.e Dunaliella, Haptophytes) Extract directly using a Potter homogeniser or a mortar and pestle to homogenise filter and algae If have a cell wall (i.e Chlorella, Tetraselmis, Nannochloropsis, diatoms) a b c d Defrost sample (if frozen) Put the filter in a 4mL glass test tube, Add mL liquid N2 and after 30 sec, Crush the filter completely with a glass rod (this is critical in order to achieve complete extraction in the later steps) GO TO EXTRACTION STEP Determine Dry weight & Ash-free dry weight 276 N.R Moheimani et al Method 16.4 Chlorophyll assay General Precautions: Work in dim light - pigments are easily bleached by lights, especially daylight and fluorescent light If using acetone add a pinch of MgCO3 to the acetone before you grind the tissue This will remove any traces of acids that may come from the algae or from the glassware Acids remove the Mg2+ from chlorophylls, forming phaeophytin Keep extracts cool at all times to prevent pigment breakdown Use chilled solvents, and grind and store algae and extracts in an ice-bath in the dark Add 5-10 mL of 90% (v/v) acetone or 100% ethanol to sample from Extraction Step of Method 16.3 Pour off supernatant (if using filtered algae) into a graduated glass centrifuge tube and, if necessary, re-extract with fresh solvent Pour off this supernatant and combine the supernatants Make sure the solvent extracts are well mixed and then centrifuge them in a bench centrifuge to get rid of any particulate matter Measure the volume of the supernatant carefully Measure absorbance at the required wavelengths (see equations below) Calculate the concentrations of chlorophyll using the equations below Note: Two different alternate sets of equations are provided The concentrations of chlorophylls a, b and c in mixtures are calculated using the equations below, where for example, E664 = the absorbance of the extract measured through cm of solution at 664 nm (Results are in µg.mL-1) Remember to adjust for dilution in your calculations if necessary (A) Equations of Jeffrey and Humphrey (1975) : a For green algae which contain chlorophylls a and b chlorophyll a = 11.93 E664 - 1.93 E647 chlorophyll b = 20.36 E647 - 5.50 E664 b For diatoms, chrysomonads and brown algae (containing chlorophyll a and chlorophylls c1, and c2 in equal proportions) USE 100% acetone as the solvent chlorophyll a = 11.47 E664 - 0.40 E630 chlorophyll c1 + c2 = 24.36 E630 - 3.73 E664 c For dinoflagellates and cryptomonads (containing chlorophylls a and c2) chlorophyll a = 11.43 E663 - 0.64 E630 chlorophyll c2 = 27.09 E630 - 3.63 E663 d For mixed phytoplankton populations (containing chlorophylls a and b, and equal amounts of chlorophylls c1 and c2) chlorophyll a = 11.85 E664 - 1.54 E647 - 0.08 E630 chlorophyll b = -5.43 E664 - 21.03 E647 - 2.66 E630 chlorophyll c1 + c2 = -1.67 E664 - 7.60 E647 - 24.52 E630 e For red algae, containing chlorophyll a only chlorophyll a = 11.41 E664 (B) Ritchie (2006 and 2008) equations for determining chlorophyll a, b, c and d: If 90% acetone is used as a solvent: chlorophyll a = -0.3319 E630 - 1.7485 E647 + 11.9442 E664 - 1.4306 E691 chlorophyll b = -1.2825 E630 + 19.8839 E647 - 4.8860 E664 -2.3416 E691 chlorophyll ctotal = 23.5902 E630 - 7.8516 E647 - 1.5214 E664 -1.7443 E691 chlorophyll d = 21.3877 E630 + 10.3739 E647 + 5.3805 E664 + 5.5309 E691 If 100% ethanol is used as a solvent: chlorophyll a = 0.0604 E630-4.5224 E647+13.2969 E664-1.7453 E691 chlorophyll b = -4.1982 E630 + 25.7205 E647 - 7.4096 E664 -2.7418 E691 chlorophyll ctotal = 28.4593 E630 - 9.9944 E647 - 1.9344 E664 -1.8093 E691 chlorophyll d = 24.1209 E630 + 11.288 E647 + 3.7620 E664 + 5.8338 )E691 16 Standard Methods for Measuring Growth of Algae and Their Composition 277 Method 16.5 Modified Bligh and Dyer and Folch methods for total lipid extraction and quantification Precautions: • • When applying this method to a new species of algae the effectiveness of extraction should be checked in preliminary experiments (see section 4) The extracts can be somewhat hygroscopic and samples must be stored over dessicant and weighing should take place in a dry atmosphere In our experience changes in atmospheric humidity can affect the results BLIGH AND DYER FOLCH Prepare fresh methanol : chloroform : deionised water 2:1:0.8 v/v/v) Add mL methanol to sample of extraction step from Method 16.3 and homogenise well Add 1mL of the solvent mixture to sample from extraction step of Method and homogenise well, and transfer from the glass test tube into a conical bottom polypropylene centrifuge tube with screw cap Add extra mL methanol to sample and mix well Wash the glass test tube with another 1mL of solvent and add to the polypropylene centrifuge tube Add mL chloroform and mix well Top up the solvent in polypropylene centrifuge tube to 5.7 mL and screw the lid tightly onto the tube Add extra mL chloroform (use to also rinse off the glass rod) and mix well for – 10 sec with vortexing Centrifuge at 1000-2000 x g for 10 or until a compact pellet is formed Centrifuge at 1000 - 2000 x g for 10 or until a compact pellet is formed Carefully transfer the supernatant to a 20 mL glass tube with screw cap (keep the lid always closed to avoid evaporation) For the second extraction, add 5.7 mL of the solvent to the pellet in the plastic centrifuge tube and close the lid, resuspend the pellet by vortexing and repeat steps and (the volume in 20 mL glass tube should be 11.4 mL) Re-extract the pellet with mL methanol:chloroform (1:2 v/v) solvent, mix well for – 10 s by vortexing and repeat steps and Add mL deionised water to the 20 mL glass tube and mix well by vortexing Add supernatant of step to the 20mL glass tube with screw cap (step 6) Add mL chloroform to the 20 mL glass tube and mix well by vortexing Add mL of deionised water and mix well by vortexing vigorously to form phase separation 10 Leave sample for 24h in the dark and ~ 5ºC for a phase separation Alternatively, samples can be centrifuged at 1000 - 2000 x g for 10 (recommend if the sample is being used for extra analysis) The top phase is the methanol/water layer and the bottom phase is the chloroform phase containing the lipids 11 Remove the methanol/water layer on the top with a very fine Pasteur pipette connected to a syringe 278 N.R Moheimani et al Method 16.5 (continued) 12 Add 6-8 drops of toluene to the chloroform layer to remove any small amount of remaining water 13 Transfer the chloroform layer to a dry and pre-weighed vial 14 Carefully remove the toluene/water on the surface of the chloroform layer in the vial 15 Immediately put the vial under a stream of ultra pure N2 gas for evaporation on a heating plate at 38ºC 16 After evaporating, keep the sample in a vacuum dessicator over KOH pellets over night and then weigh the vial using a digit balance Method 16.6 Nile Red lipid visualization method (Dempster and Sommerfeld 1998) Prepare a fresh Nile Red stock solution (100 µg.mL-1) by adding 10 mg Nile red in 100 mL acetone Add 12.5 µL of stock solution to mL fresh algae culture View the cells under compound microscope using UV light (i.e UV filter 460-490 nm) NB Samples must be viewed quickly as long term exposure to UV will result in degradation of dye 16 Standard Methods for Measuring Growth of Algae and Their Composition Method 16.7 Thin layer chromatography of total lipids from microalgae (TLC) Precautions: All work should be carried out in a flame-proof fume hood Note that Iodine vapour is toxic A TLC Plate activation and chamber preparation o Pre-heat oven at 120 C Verify running direction of the plate by looking at the production marks on the aluminium back of the TLC plate (e.g aluminium-backed TLC plates (Merck TLC Silica Gel 60F254)) Orient the plate such that production marks are vertical and draw a faint baseline and reference line 10 mm each from the plate edge with a soft (2B) pencil and clean ruler Place the plate upright in between clean and grease-free glass plates in the carrier to prevent warping Activate (dry) the plate for 1h at 120°C After activation, cool the plate down in a moisture-free environment while keeping the plate sandwiched between the glass plates Prepare developing chamber in a fumehood ensure that it is free of any dust, chemicals and solvents other than those used for the chromatographic run Add in the freshly prepared solvent mixture Place the filter paper in the chamber and allow the solvent vapor to equilibrate in the chamber for ~1 h B Sample loading and TLC run After total lipid extraction (Method 16.5) fully dissolve the dried lipid sample in vials in 20 µL of chloroform for loading the TLC plate 10 While waiting for TLC tank to be saturated, load the activated TLC plate with the lipid sample 11 Pipette 20 µL of lipids dissolved in chloroform and apply as a discrete spot on the baseline and allow the chloroform to evaporate completely 12 Quickly transfer the loaded TLC plate in the saturated developing chamber 13 Allow the chromatographic process to run undisturbed for approx 50 or until the solvent front has reached the reference line c Derivatisation and lipid class identification 14 Remove the plate from the developing chamber and allow to dry for approx 30 in a fume hood 15 Carefully transfer the plate to the iodine chamber and allow for the derivatisation process to complete until all spots on the TLC plate are clearly revealed 16 Carefully outline the visible spots with a pencil and calculate the Rf value of each spot by using the equation: distance of the spot from baseline Rf = distance between baseline and reference line 17 Identify the groups of lipids by comparing calculated Rf values with those published in the literature where a similar solvent system has been used or comparison with a standard lipid (oil) run on the same system 18 Derivatised plate can be scanned and saved electronically using a scanner 279 280 N.R Moheimani et al Method 16.8 External hydrocarbon measurement method (Adapted from Eroglu and Melis 2010) Prepare a sample of ~1 g wet cell weight (approximately 0.2 g AFDW) of Botryococcus braunii by filtration or centrifugation Mix the sample with g of glass beads and 10-20 mL of n-heptane (alternatively hexane) for 1-3 Add 10-15 mL of growth medium to the mixture resulting in two phase partition - the aqueous phase will contain cells and solvent phase will contain external B braunii hydrocarbons Remove the solvent the layer and measure absorbance at 190 nm (UV-spectrophotometer) against a squalene standard curve) 16 Standard Methods for Measuring Growth of Algae and Their Composition 281 Method 16.9 Total carbohydrate determination Precautions : • When applying this method to a new species of algae the effectiveness of extraction should be checked in preliminary experiments (see section 4) • This method uses concentrated acid and phenol Appropriate safety equipment must be worn Reagents Glucose standard solution: 0.1 g L-1 Phenol stock solution: 50 g L-1 1M H2SO4 Concentrated H2SO4 Carbohydrate Standard curve Prepare carbohydrate standard curve (see bottom of this flowchart) in acid resistant test tubes Assay method Homogenise sample- from extraction step of Method 16.3 in 0.5 mL 1M H2SO4 in a 10 mL acid resistant plastic test tube with screw lid A fresh standard curve must be prepared for each set of samples Top up to mL with 1M H2SO4 Tighten the lid and incubate in a 100°C water bath for 60 Cool to room temperature (~30min) and centrifuge at 1000 – 2000 x g for 5-10 Pipette mL of the supernatant into another acid resistant test tube In fume hood, add mL of phenol solution and rapidly mix well using a Vortex stirrer In fume hood, rapidly add mL concentrated H4SO2 (Notes : (a) To avoid splashing acid, keep the pipette tip in a light angle against the test tube wall, (b) acid resistant protective equipment must be worn) Close the test tube lid (tighten very well) and mix well by vortexing Cool the test tube for 30 at room temp 10 Mix the samples well manually 11 Read the absorbance at 485 nm and calculate the carbohydrate content from the standard curve using the equation: Carbohydrate yield (mg.L-1) = Carbohydrate value from standard curve volume of digested material × culture volume Suggested Glucose Standard Curve Glucose (µg)* 40 80 120 160 200 Standard glucose solution (mL) 0.4 0.8 1.2 1.6 dH2O (mL) 0.16 1.2 0.8 0.4 * If the standard curve is NOT linear use a lower range of concentrations 282 N.R Moheimani et al Method 16.10 Total protein determination methods Precaution: When applying this method to a new species of algae the effectiveness of extraction should be checked in preliminary experiments (see section 4) Reagents Bovine serum albumin (BSA) Fraction V -stock solution: 2.5 BSA L-1 Stock solutions for Biuret reagent (a) Na2CO3 = 200 g.L-1 (b) NaOH = 40 g.L-1 (c) NaK tartrate = 200 g.L-1 (d) CuSO4.4H2O = 50 g.L-1 Biuret reagent preparation: Using the stock solutions above, add 20 mL of (a) + 20 mL of (b) + 160 mL of deionised water, mix well and then add 2mL of (c) and mL of (d) Folin-phenol reagent preparation: Dilute Folin reagent 1:1 with deionised water Protein standard curve Lowrey et al method Prepare protein standards in 10 mL centrifuge tubes in triplicate (see bottom of this flowchart) Add of 1mL Biuret reagent to sample from extraction step of Method 16.3 and mix well with a glass rod After mixing carefully transfer the contents to a 10 mL centrifuge tube Grind the samples from extraction step of Method 16.3 in mL of phosphate buffer (pH 7.6) After mixing carefully transfer the contents to a 10 mL centrifuge tube Pour another 1mL Biuret reagent over the glass rod into the 4mL glass tube and, mix well, Centrifuge homogenate at ~1500 x g for 20 Transfer the content to a 10 mL centrifuge tube Pour supernatant to a separate test tube Add extra mL Biuret reagent to the 10mL centrifuge tube The volume of all of the samples in tubes the needs to be made equal by adding phosphate buffer solution and the extraction can be stored at 4°C for further analysis Bradford method A fresh standard curve must be prepared for each set of samples Add mL Biuret reagent to each centrifuge tube Transfer 30 µL of extracted sample and standards to separate tubes and mix with 70 µL distilled water Place the sample and protein standard tubes in a 100ºC water bath for 60 Place clean glass marbles over the opening of the centrifuge tubes to prevent losses from splattering Add 2.9 mL of Coomassie Brilliant Blue solution to samples and standard and mixed thoroughly (total volume mL) Remove the tubes from the water bath and immediately add 0.5mL Folin-phenol reagent while mixing in a vortex stirrer* *Note: Since the Folin phenol reagent breaks down rapidly in the alkaline solution of the samples, rapid mixing while adding it is essential Place tubes in a 10-15°C water bath for 20 and then allow to equilibrate to room temperature for another 15 Incubate all samples for at room temperature Measure absorbance of samples and standards at 600 (595) nm against the reagent blank 16 Standard Methods for Measuring Growth of Algae and Their Composition 283 Method 16.10 (continued) Centrifuge at 1000-2000 x g for 5-10 10 Remove the supernatant carefully and read the absorbance at 660 nm 11 Determine the protein content of the samples using a standard curve from protein standard using the equation: Protein value from standard curve volume of digested material ⫻ culture volume Suggested Protein Standard Curve Protein (µg) 50 100 150 200 250 300 350 BSA V (mL) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 dH2O (mL) 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 References Ackman RG (1991) Application of thin-layer chromatography to lipid separation: neutral lipids In: Perkins EG (ed) Analyses of fats, oils and lipoproteins American Oil Chemists Society, Champaign, pp 60–82 Ackman RG, McLeod CA, Banerjee AK (1990) An overview of analyses by Chromarod-Iatroscan TLC-FID J Planar Chromatogr 3:450462 Barbarino E, Lourenỗo S (2005) An evaluation of methods 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and lipid composition of 10 species of microalgae used in mariculture J Exp Mar Biol Ecol 128:219–240 Zhu CJ, Lee YK (1997) Determination of biomass dry weight of marine microalgae J Appl Phycol 9:189–194 Index A Absorbance, 267–269, 276, 280–283 Adequate mixing, 117, 119 Aerobic wastewater treatment, 156 Agmenellum, 218 Airlift, 5, 81, 117, 122–124, 136, 227, 228 Alexandrium, 82 Algae Species Programme, 7–8 Algal grazers, 155–156 Alternative fuels, 99 Amphidinium, 105 Anabaena, 50, 61, 62, 95, 147 Anaerobic digestion, 145, 147, 154, 158–161, 234, 235, 238, 242, 244–248, 250–253 Arthrospira, 6, 61, 81, 116, 133, 134, 137, 142–146, 148, 171, 176, 218 Asexual lifecycles, 99 Ash free dry weight (AFDW), 146, 147, 165, 257, 267, 269, 271, 274, 275, 280 Autoflocculation, 168, 170 Autoflotation, 175 Autoinhibitor, 4, 84, 145 B Betaine lipids, 20–21, 27, 29, 31 Bicarbonate, 25, 81, 94, 137, 144, 154 Biobutanol, 77 Biocoil, 9, 10, 260 Biodiesel, 21, 77, 91, 123, 154, 184, 200, 208, 231, 233, 256 Biodiesel standards, 207–214, 218, 219 Bioethanol, 77, 81, 115, 159, 190 Bioflocculation, 109, 157, 158, 168, 170 BioH2, 106 Bioprospecting, 77 Biosynthesis, 17, 21, 24–28, 30–33, 39, 41, 43–45, 48, 49, 52–53, 103, 108 Bligh and Dyer, 78, 193–196, 201, 272, 277 Botryococcene, 9, 21, 24 Botryococcus braunii, 9, 21, 22, 24, 29, 81, 82, 121, 191, 213, 258, 272, 280 b2TUB (b2-tubulin), 102 By-products, 17, 204, 259–261 C Canola, 207, 234–236, 241, 242, 245–247, 250, 252 Capital expenses (CapEx), 158, 256, 258 Carbohydrate, 20, 37, 57, 59, 62, 115, 128, 158, 159, 203, 204, 224, 271, 272, 281 Carbon acquisition, 91, 95–96 Carbon concentrating mechanisms (CCM), 95, 96 Carbon dioxide (CO2), 8, 17, 20, 64, 119, 137, 234, 237, 238, 240–246, 255 Carboxylase, 4, 24, 25, 56, 60, 80, 91, 95, 106, 224 Carotenoids, 7, 17, 81, 104, 106–108, 160, 190, 194, 203, 204, 260 Cell counting, 267–268, 273 size, 80, 84, 91, 157, 268 wall, 80, 81, 84, 100, 102, 104, 158, 167, 187, 190–192, 200, 238, 271, 275 Cellular lipid, 32, 92, 187, 194 Centrifugation, 6, 10, 157, 171, 173–174, 178, 182, 192, 199, 201, 230, 240, 241, 243, 245, 247, 252, 269, 280 Cetane number, 200, 210–211, 213, 218 Chaetoceros, 29, 31, 80, 81, 214 Charge, 19, 94, 128, 157, 166–171 Chemoorganotrophy, 91 Chladophora sp, 29 Chlamydomonas, 4, 19, 20, 30, 93, 100, 101, 104 Chlamydomonas reinhardtii, 20, 21, 27–32, 39–41, 43, 53, 55–60, 63, 94, 99–108 Chlorella, 2, 4, 6, 7, 9, 19, 30, 31, 54, 78, 81, 104, 107, 134, 135, 139, 143–148, 154, 194, 195, 215, 237, 258, 260, 271 Chlorella pyrenoidosa, 2, Chlorella vulgaris, 1, 26, 29, 32, 82, 104, 105, 193, 213–215 Chloroccoccum, 32 Chlorococcum littorale, 8, 80, 139 Chlorophyll, 20, 21, 57, 64, 78, 92, 93, 105–107, 139, 156, 194, 267–269, 271, 276 Chlorophyll determination, 271 Chloroplast, 21, 24, 26–28, 30–32, 57, 59, 60, 99–103, 108, 189, 191, 194 membranes, 20, 27, 100, 189, 191 transfomration, 100, 105 Chondria armata, 20 Circular central pivot ponds, 133, 135, 147 Circular ponds, 3, 5, 116, 135 Closed photobioreactor, 2, 8–10, 79, 81, 84, 128, 137, 138, 144, 146, 153, 223, 225, 234, 270 Clostridium leptum, 41 Clostridium methylpenosum, 41 Clostridium pasteurianum, 41, 43 Cloud point, 210, 212, 213, 218 C:N ratio, 154 Coagulation and flocculation, 167–171 CO2 diffusion, 80 Cold flow, 207, 210, 212–214, 218, 219 Commercialization, 7, 255 Commercial production, 6–7, 79, 83, 107, 115, 116, 119, 128, 135, 237 Competitive strains, 81 M.A Borowitzka and N.R Moheimani (eds.), Algae for Biofuels and Energy, Developments in Applied Phycology 5, DOI 10.1007/978-94-007-5479-9, © Springer Science+Business Media Dordrecht 2013 285 286 Concentration factor, 165–167, 169, 175, 177, 178, 180–182, 199 Constraints on photoautotrophic growth, 91 Co-products, 28, 81, 108, 115, 153, 203, 234, 236, 239, 242–244, 249, 250, 253, 255, 256, 259–261 Covalent association, 192 Crocosphaera, 50 Crypthecodinium cohnii, Cryptomonas, 29, 214 Cultivation method, 223 Culture maintenance, 82, 85 Cyanobacteria, 20, 26, 29, 37, 38, 46, 50, 52, 53, 60–64, 95, 96, 115 Cyanothece, 50, 61 Cyclotella, 147 Cylindrotheca, 119 D Dewatering, 10, 80, 133, 160, 165–184, 235, 241, 243, 252, 255, 257, 258 Diacylglycerol, 22, 26–28 Diatom, 1, 2, 18, 19, 26, 28–31, 82, 85, 105, 106, 125, 197, 199, 201, 269 Dinoflagellates, 30, 81, 82, 95, 104, 105, 218, 276 Diphosphatidylglycerol (DPG), 18 Dissolved air flotation (DAF), 157, 169, 174, 175, 241 Dissolved inorganic carbon (DIC), 94–96 Docosahexaenoic acid (DHA), 17, 29, 31, 160, 187, 212, 260 Doubling time, 17, 62, 269 Downstream processing, 10, 79, 81, 83, 165, 224, 261 Drying, 6, 10, 122, 159, 166, 179, 181, 192, 198, 230, 243, 260 Dry weight, 2, 21, 31, 32, 61, 62, 82, 139, 142, 146–148 156, 160, 238, 240, 258, 267, 269, 271, 274, 275 Dunaliella, 7, 30, 80, 81, 83, 94, 116, 135, 145, 148, 181, 214, 215, 268, 271, 275 Dunaliella salina, 5, 7, 28, 30, 32, 77, 81, 83, 85, 104, 105, 107, 133, 134, 137, 144, 147, 191, 239, 257, 260 E Eicosapentaenoic acid (EPA), 19, 20, 27, 29, 31, 32, 82, 160, 187, 212, 217, 219 Electrocoagulation, 168, 170–171 Electroflocculation, 171 Emilianea, 82 Energy, 1, 17, 37, 77, 94, 107, 115, 137, 153, 165, 187, 207, 223, 234, 255 Ethanol, 8, 40, 56, 60, 107, 115, 125, 128, 154, 158, 159, 192, 194–200, 230, 237, 258, 271, 273, 276 Euglena, 18 Extensive ponds (lagoons), 116 Extraction, 9, 52, 78, 115, 158, 187, 209, 234, 255, 269 F Fatty acid profile, 28, 33, 196, 200, 201, 207–211, 213–219 Fatty acids, 17, 77, 99, 115, 145, 159, 187, 208, 260, 272 [FeFe]-Hydrogenase, 38–45, 47, 48, 53–56, 58, 63 FeMo-cofactor, 51–53 Fermentation, 4, 7, 8, 43, 46, 53, 54, 58, 60, 62, 64, 65, 107, 158, 159, 190 Fermenters, 116 Filter press, 176, 179, 181 Filtration, 80, 133, 145, 157, 165, 171, 175–182, 184, 193, 194, 198, 199, 201, 203, 212, 230, 269, 271, 275, 280 Index Flashing light effect, 4, 224, 226, 227 Flat panel photobioreactor, 8, 9, 61 Flocculation, 145, 157, 167–171, 174, 181, 182, 184, 235, 240, 241, 244–249, 252 Flow cytometry, 77, 83, 268–269 Flue gas, 9, 17, 123–127, 137, 138, 154, 237, 241, 245 Folch, 78, 277 Fossil fuels, 7, 99, 115, 116, 125, 127, 153, 157, 158, 160, 161, 231, 234, 237, 245, 247, 252 Froth flotation, 10 Fuel properties, 207–219 G Galdieria, Genetic diversity, 49–50 Genetic engineering, 8, 85, 99–109 Genetic transformation, 99–101, 105 Genome, 28, 38–41, 46, 50, 55, 60–62, 82, 92, 100–103, 105 Genomics, 60, 91 Genomic sequence, 100 Geranylgeranyl-pyrophosphate (GGPP) synthase enzyme, 108 Global warming potential (GWP), 237 Glycerol, 18–20, 26, 27, 30, 32, 60, 208, 242–244 Glycolipids, 19, 20, 31, 78, 187, 190, 194, 203 Glycosylglycerides, 17, 19–20, 27, 29 Gravity belt filter, 178 thickeners, 172 Growth rate, 28, 78, 82, 91, 92, 96, 99, 138, 139, 225, 226, 238, 239, 248, 249, 252, 257, 268–271 GTPase, 44, 49 H H2, 64 Haematococcus, 5, 7, 80, 191, 226, 271 Halophilic, 7, 32, 77, 137, 275 Halothermothrix orenii, 40 Harvesting, 6, 61, 80, 93, 99, 115, 133, 157, 165, 187, 223, 234, 255 HCO3-, 79, 94–96, 137 Helical photobioreactor, Heterotetrameric, 51 Heterotrophic respiration, 37 High cell density cultures, 8, 139 High light, 29, 61, 64, 92–94, 96, 107, 108, 135, 137, 139, 142, 224, 227 High pressure extraction, 234, 243 High rate algal pond (HRAP), 153–161 History, 1–10, 51, 194 Homogenization, 191, 192, 195 Homologous recombination, 85, 100–104, 106 Homoplasmic lines, 101 HSP70A, 102, 103 HydA, 39–41, 44, 45, 58 Hydrocarbon, 9, 17, 21–24, 77, 81, 82, 156, 158, 187, 188, 192, 194, 211–213, 230, 238, 258, 272, 280 Hydrogen, 4, 9, 37, 38, 47, 48, 51–53, 55–62, 77, 106–107, 171, 230 Hydrogenase, 37–65, 106, 107 Hydrogen bonding, 189, 192 production pathway, 55–56 Hydrophobic, 18, 46, 48, 63, 181, 192, 193, 230, 272 Index I Inclined (cascade) systems, 133–134 Inoculum, 2, 3, 83, 85, 119, 141, 144, 145, 245 Inorganic carbon, 32, 40, 79, 94–96, 137 Inorganic coagulation and flocculation, 169 Irradiance, 8, 29, 62, 80, 83, 84, 86, 93, 94, 121, 124–126, 139–142, 146, 194, 224, 227, 258, 259, 265–267 Isochrysis, 9, 214 Isoprenoid hydrocarbons, 21 K Kinematic viscosity, 210, 211, 218 L LHCB1, 102 Life cycle assessment (LCA), 184, 234–238, 242, 243, 245, 252 Light, 4, 20, 41, 78, 91, 99, 116, 135, 197, 212, 223, 258, 265 Light:dark cycle, 4, 29, 30, 54, 61, 92, 117, 141, 143, 225, 226 Light harvesting, 20, 64, 85, 107 Light harvesting antenna, 57, 93, 107 Light limitation, 93, 96 Linear electro-dewatering, 179–180 Lipid, 2, 17, 77, 91, 99, 119, 138, 158, 187, 207, 224, 240, 256, 269 fractionation, 187, 193, 194 metabolism, 17, 24, 28, 29, 32 productivity, 2, 28, 31, 32, 78–80, 83–85, 119, 127, 145, 259 Lithotrophy, 91, 92 Lubricity, 207, 213 Lycopadiene, 21 M Mass transfer capacity, 117, 119 Measuring growth, 265–283 Metabolic engineering, 60, 65, 103, 104, 106–108 Metabolism, 17–33, 37, 38, 43, 45, 52–61, 64, 92, 95, 194, 224 Methane, 4, 7, 46, 64, 77, 120, 158–160, 234, 237, 242, 245, 246, 259, 260 MicroRNA (miRNA), 103, 104 Mixed pond, 133, 135, 139 Monodus, 143 Monogalactosyldiacylglycerol (MGDG), 19, 22, 26, 27, 29, 31, 32, 187, 194 Monoraphidium, 79, 197 Morphology, 80–82, 84, 165, 267 mmax, 91, 92 N Nannochloris, 31 Nannochloropsis, 23, 29, 80–82, 85, 93, 94, 119, 122, 126, 137, 144, 191, 192, 213–215, 219, 238, 240, 258, 260, 271, 275 Navicula, 219 Neocallimastix frontalis, 41 Neubauer haemocytometer, 267, 268, 273 N2 fixation, 37, 46, 51–53, 61 nif, 50 [NiFe]-hydrogenases, 38, 40, 43, 45–49, 59, 62 Night time temperature, 79, 142 Nile Red, 78, 272, 278 NIT1 (NIA1), 102, 103 Nitrogenase, 37–65 Nitrogen limitation, 21, 30, 31, 159 287 Non-polar lipid, 17, 24, 29, 189, 192, 193 Nuclear transformation, 102, 104–106 Nutrient removal, 153, 154, 156, 157, 160 Nutrients, 17, 24, 30–32, 61, 80, 83, 95, 109, 115, 121, 124, 127, 137, 139, 144, 145, 153, 156, 158, 160, 161, 166, 226, 238, 240, 241, 259, 260 O Ochromonas danica, 19, 191 Oil extraction, 126, 158, 209, 234, 235, 238, 241, 242, 245, 251, 252, 259 Operating expenses (OpEx), 158, 256, 258 Optimum temperature, 79, 82, 139, 141, 143 Organic coagulation and flocculation, 169 Organotrophy, 91, 92 Oscillatoria, 61, 218 Osteococcus tauri, 28, 82 Oxidative stability, 207, 210–214, 218, 219 Oxygenase, 80, 91, 95, 140 Oxygen sensitivity, 48, 58–59, 106 tolerance, 79–80 P Paddle wheel, 5, 116, 127, 134, 136–139 Parietochloris, 21–23, 29, 213, 214, 216 Particle size, 166, 171–173, 178, 183, 230 Pathogens, 84, 155–156 Pavlova lutheri, 19, 22, 23, 27, 29, 31 pH, 2, 8, 19, 20, 30, 57, 61, 62, 79–81, 94, 116, 117, 119–121, 126, 137, 144, 154–157, 166–170, 175, 181, 192, 196, 197, 199, 226, 229, 269, 275, 282 Phaeodactylum, 137, 147, 219, 240 Phaeodactylum tricornutum, 5, 9, 23, 28, 29, 82, 94, 105, 190, 192, 197, 198, 200, 218, 228, 239, 259, 261 Phosphate starvation, 32 Phosphatidylcholine (PC), 18, 20, 22, 26, 27, 29–32, 187 Phosphatidylethanolamine (PE), 18, 22, 28, 30, 32, 187 Phosphatidylglycerol (PG), 18, 20–22, 26, 28–31, 187 Phosphatidylinositol (PI), 18, 22, 27, 30, 187, 201 Phosphatidylserine (PS), 18, 30, 187 Phosphoglycerides, 17–19, 26, 27 Phospholipids, 18, 29–32, 78, 187, 190, 193, 194, 203 Photoacclimation, 140, 141 Photoinhibition, 80, 84, 85, 93, 107, 116, 128, 140, 143, 224 Photolithotrophy, 92 Photosynthesis, 4, 8, 10, 20–21, 24, 28, 37, 38, 52, 55, 57, 58, 61, 64, 79, 80, 84, 91, 93–96, 99, 100, 121, 137–140, 143, 146, 153, 154, 157, 160, 223, 224, 258, 265, 266 Photosynthetically active radiation (PAR), 29, 62, 63, 92, 157, 224, 258, 265–267 Photosynthetic efficiency, 2, 8, 64, 84, 116, 123, 124, 138, 139, 146, 258 Phycology, 1, 2, 4, 265, 267 Pilot scale, 10, 118, 119, 122, 125, 154, 156, 183, 227, 259–261 Plasma membrane, 18, 100, 187, 189–191 Plastid transformation, 105, 106 Pleurochrysis, 81 Polarity, 187, 189, 193, 194, 196, 197, 201, 258 Polar lipid, 17, 19, 21, 30, 31, 187–190, 192–194, 196, 197 Polyunsaturated fatty acids, 17, 19–21, 27–29, 77, 81, 115, 159, 160, 187, 199, 200, 209, 212, 214, 218, 219, 260, 261 Porphyridium, 5, 105, 106, 260, 268 288 Production cost, 7, 79, 108, 118, 122, 133, 255, 257, 259–261 Prorocentrum, 30 Protein, 2, 17, 39, 80, 91, 99, 115, 158, 189, 243, 260, 271 Protococcus viridis, Protozoa, 2–4, 20, 39, 40, 81, 85, 143, 144 PSAD, 103 Pyrococcus furiosus, 46, 47 Pyrolysis, 77, 158, 258 R Raman spectroscopy, 78 Rapeseed, 26, 207 Rapid screening, 78, 83–86, 272 RBCS2, 102–104 Recovery efficiency, 165, 167, 169, 178 Recycling the medium, 2, 84, 127, 145, 175 Renewable energy, 1, 10, 37, 122, 125, 208, 250 Research Institute of Innovative Technology for the Earth (RITE), 8, 10 Respiration, 28, 37, 38, 45, 47, 55, 57–59, 79, 80, 139, 142, 224, 230 Respiration rate, 57, 79, 80, 84, 139, 142, 143 Rhodomonas, 29, 214 Rhodospirillum rubrum, 47 Riboswitches, 106 Rubisco, 56, 57, 63, 80, 84, 91, 92, 95–96, 102, 140 S Salinity, 22, 61, 79–84, 116, 133, 137, 144, 158, 166–168, 171, 181, 239, 259, 269 Saponification, 188, 190, 194–200, 203 Scale-up, 184 Scenedesmus, 4, 5, 19, 24, 32, 80, 119, 134, 137, 147, 157, 177, 192, 216, 260, 271 Schizochytrium, 214 Secondary metabolites, 92 Sedimentation, 136, 157, 165, 171–173, 175, 181, 182, 184, 230 Selection, 77, 79–83, 85, 86, 100, 101, 103, 104, 109, 116, 141, 155, 157, 181–184, 187–190, 258 Selenastrum, 29 SimaPro software, 237 SlyD protein, 49 Solid liquid separation, 165–168, 171, 172, 226, 230 Solvent extraction, 78, 187–204, 234, 241, 244–248, 252 Sonication, 81, 191, 192, 258, 271 Specific growth rate, 78, 91, 92, 138, 139, 257, 269–271 Spirulina, 5, 6, 9, 10, 61, 80–82, 94, 133–137, 141–148, 176, 181, 196, 216, 218, 238, 260 SQDG, 19, 20, 22, 26, 29, 31, 187 Squalene, 21, 24, 280 Stephanodiscus, 31 Sterols, 17, 78, 187, 193, 194, 203, 261 Storage lipids, 21–24, 30, 31, 108, 194 Index Strain selection, 77–86, 128 Sulfur deprivation, 57–58, 63, 107 Surface volume ratio, 116, 227 Suspended air flotation (SRF), 175 Symbiodinium, 105, 218 Synechococcus, 46, 50, 62 T Tangential (cross) flow filtration (TFF), 176–178 Techno-economic modelling, 255–257 Temperature, 4, 5, 8, 9, 21, 28–30, 48, 61, 78–84, 108, 116–120, 126, 135, 137–143, 146, 155, 158, 166, 168, 170, 172, 187–197, 199–201, 208, 211, 212, 214, 224–226, 229, 230, 240, 259, 281, 282 Tetraselmis, 9, 31, 38, 122, 147, 171, 192, 216, 258, 271, 275 Thalassiosira, 28, 30, 82, 105, 106 Thickening, 157, 165–184, 258 Tichocarpus crinitus, 29 Tissue grinder, 191 Transcriptional regulation, 58, 62 Transformant, 101–104 Transgene, 100–103, 107 Transgene expression, 100, 102, 103 Triacylglycerols, 17, 21, 22, 77, 78, 119, 192, 207–209 Trichomonas vaginalis, 39–41 Triterpenoid hydrocarbons, 24 Trophic conversion, 107 Tubular photobioreactors, 7, 9, 10, 116, 122–124, 126, 137, 142, 190, 194, 202, 226, 258, 259, 261 Turbulence, 62, 94, 117, 118, 127, 128, 134, 138, 172, 224, 226 U Ultrasound, 168, 170, 191, 192 Unsaponifiable constituents, 187, 197, 203 Using wastewater, 22 V Vacuum filters, 75, 77, 179 vnf, 50 W Wastewater treatment, 4, 5, 8, 10, 65, 77, 115, 133, 135, 145, 153–161, 168, 169, 174, 180, 181, 238, 259, 261 Water jets, 134, 136–137 Wax esters, 17, 187, 194 Wild type strains, 83, 108 Z Zeta-potential, 166