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E-raamat: Protocols for Macroalgae Research

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  • Ilmumisaeg: 17-Apr-2018
  • Kirjastus: CRC Press Inc
  • Keel: eng
  • ISBN-13: 9781351132947
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  • Formaat: PDF+DRM
  • Ilmumisaeg: 17-Apr-2018
  • Kirjastus: CRC Press Inc
  • Keel: eng
  • ISBN-13: 9781351132947

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This book presents a wide range of tested and proven protocols relevant to a number of fields within biotechnology used in laboratory experiments in everyday phycological (seaweed) research. A major focus will be on bioenergy related aspects of this emerging technology. These protocols will be written in a clear and concise manner using simple language permitting even nonspecialist to adequately understand the significance of this research. It will also contain all necesssary notes and guidelines for successful execution of these experiments.

Arvustused

"This book is an impressive picture of the tremendous evolution of phycology during the ten past years. Each PhD student and postdoctoral researcher should have it in its laboratory; he will find in it the basic most recent protocols ready to use for his study, and the matter for imaginative, limitless experiments." Bruno de Reviers, Muséum National dhistoire Naturelle, Paris, France

""This book is a well written and informative guide to methods for macroalgal (seaweed) culture and analysis. It is notable for the broad coverage of macroalgal biology, from culture approaches, through methods for compositional analysis to the newer molecular approaches of metabolomics and transcriptomics. It will certainly prove to be a useful resource for those involved in macroalgal culture and biotechnology."John Beardall, Monash University, Melbourne, Victoria, Australia

Protocols for Macroalgae Research describes state-of-the-art approaches and applications in seaweed research. The book is comprised of 31 protocols in all and provides critical information regarding the cultivation and preservation of seaweeds, their chemical composition, as well as their cellular and molecular characterization. This type of research is critical and timely, as the last few decades have witnessed remarkable advancements in phycological research worldwide. Consideration of algal resources for food, fuel, chemicals, feed, and fertilizer has furthered the development of diverse laboratory techniques that not only advance their utilization substantially but also provided new insights in understanding the molecular basis of their form and function. This recent progress has enabled researchers to determine and quantify various metabolites and organic macromolecules such as carbohydrates, proteins, and lipids.

Protocols for Macroalgae Research thus uses macroalgae as a model organism for addressing fundamental research problems. No recent comprehensive literature of this format exists on this topic. As such, this state-of-the-art protocol text both develops and standardizes the various macroalgae approaches located within the field. It does so by featuring a wide-range of protocols from invited experts; each protocol features a brief introductory overview, an inventory of materials and reagents, and step-by-step experimental procedures which include helpful notes offering care and cautions. This protocol book is an invaluable reference for beginning and preexisting researchers within the field of macroalgae. "This book is an impressive picture of the tremendous evolution of phycology during the ten past years. Each PhD student and postdoctoral researcher should have it in its laboratory; he will find in it the basic most recent protocols ready to use for his study, and the matter for imaginative, limitless experiments." Bruno de Reviers, Muséum National dhistoire Naturelle, Paris, France

"There have been prior textbooks on methods in phycology, but this book brings techniques up to date. The book seeks to aid the reader to actually be able to replicate research protocols. An array of subject matter experts has been accumulated to produce 31chapters. The book is logically laid out with the reader in mind, explains techniques, and provides best-practice advice on how to carry out protocols successfully." Alan T Critchley, Cape Breton University, Nova Scotia, Canada

"This book is a well written and informative guide to methods for macroalgal (seaweed) culture and analysis. It is notable for the broad coverage of macroalgal biology, from culture approaches, through methods for compositional analysis to the newer molecular approaches of metabolomics and transcriptomics. It will certainly prove to be a useful resource for those involved in macroalgal culture and biotechnology."John Beardall, Monash University, Melbourne, Victoria, Australia

"The book covers a wide range of topics: From large-scale macroalgal cultivation to the biochemical and molecular characterization. I can see that each of the individual chapters has been written by leading experts in the respective field. I am very happy about the approach to present theoretical background first and then provide step-by-step protocols to help implementing methods in lab routines This book represents a significant enrichment to the lab work of each phycology scholar. My PhD students will be very happy with it!" Kai Bischof, University of Bremen, Germany

"A well-balanced selection of topics covering all main subjects relating to 21th century phycology (genetics development aquaculture). This is what makes this book quite unique and given the interest in seaweed for aquaculture in the western part of the world, I think this book will be warmly received."Olivier De Clerck, Ghent University, Belgium

"A really useful new addition to the literature on algae and their uses, with detailed methods for the study of these organisms, which have considerable potential in the blue economy. Thirty-one chapters in three sections (cultivating and preserving seaweeds: chemical composition; cellular and molecular characterization), by experts in their fields. Research oriented, with detailed explanations on how new techniques can be applied to the burgeoning new industries based on algal materials. Covers a wide variety of detailed methods, from culture (from commercial aquaculture to bacteria-free algal studies); through chemical constituents and health aspects, molecular techniques (including cloning and transformation); as well as detailed modern microscopic techniques on macroalgae and their related bacterial flora."John J Bolton, University of Cape Town, South Africa

"A timely, well produced book containing state of the art information on a range of methodologies relevant to seaweed scientists. It fills a large gap in the available literature and will be an essential resource for both budding and experienced macroalgal researchers."Catriona Hurd, University of Tasmania, Hobart, Australia

Preface ix
Editors xiii
Contributors xv
Section I: Cultivating and preserving seaweeds
Chapter 1 Seaweed in high-energy environments: Protocol to move Saccharina cultivation offshore
3(34)
Bela H. Buck
Britta Grote
1.1 Introduction to offshore aquaculture
4(1)
1.2 State of the art of offshore seaweed cultivation
5(3)
1.3 Land-based preparation and site selection
8(12)
1.3.1 Site selection
9(1)
1.3.1.1 Site selection: Advance information of the local area ashore and at sea
10(1)
1.3.1.2 Site selection: Site-specific, oceanographic, and water quality parameter
11(1)
1.3.1.3 Site selection: Economic/technical/expansion feasibility
12(1)
1.3.2 Equipment and system design
13(1)
1.3.2.1 System design for the offshore seaweed farm
17(1)
1.3.3 Seeding procedure
18(2)
1.4 Transfer at sea
20(9)
1.4.1 Deployment of the offshore farm
20(1)
1.4.1.1 Deployment of the farm
21(3)
1.4.2 Operation, maintenance, and harvest
24(1)
1.4.2.1 Operation and maintenance at sea
24(1)
1.4.2.2 Harvest
25(1)
1.4.3 Problems and failures
26(1)
1.4.3.1 Prevention of mistakes
27(2)
1.5 Multi-use of offshore installations
29(3)
References
32(5)
Chapter 2 Cultivation protocol for Saccharina latissima
37(24)
Silje Forbord
Kristine Braaten Steinhovden
Kaia Kjolbo Rod
Aleksander Handa
Jorunn Skjermo
2.1 Introduction
38(3)
2.1.1 The life cycle of laminariales
39(2)
2.2 State of the art
41(1)
2.3 Materials
41(2)
2.3.1 Collection of sporophytes and induction of sori
41(1)
2.3.2 Sori disinfection, dehydration, spore release, and spore counting
42(1)
2.3.3 Starting gametophyte cultures
42(1)
2.3.4 Maintenance of gametophyte cultures
42(1)
2.3.5 Fertility test
43(1)
2.3.6 Seeding
43(1)
2.4 Experimental procedures
43(13)
2.4.1 Seed supply and sporulation
43(1)
2.4.1.1 Collection of sporophytes and induction of sorus
43(1)
2.4.1.2 Sorus disinfection
45(1)
2.4.1.3 Dehydration
45(1)
2.4.1.4 Spore release
45(1)
2.4.1.5 Spore density
46(1)
2.4.2 Gametophyte cultivation
46(1)
2.4.2.1 Starting-up gametophyte cultures
46(1)
2.4.2.2 Maintenance of gametophyte cultures
48(1)
2.4.2.3 Contamination control
49(1)
2.4.2.4 Fertility test
49(1)
2.4.3 Seeding
49(1)
2.4.3.1 Seeding with spores
50(1)
2.4.3.2 Seeding with gametophytes
50(1)
2.4.3.3 Hatchery systems
50(1)
2.4.3.4 Cultivation under controlled laboratory conditions
51(1)
2.4.3.5 Development of seedlings under optimal conditions
52(1)
2.4.4 Deployment
53(1)
2.4.4.1 Transport
53(1)
2.4.4.2 Deployment at sea
53(3)
2.5 Notes
56(1)
References
57(4)
Chapter 3 Derivation of clonal stock cultures and hybridization of kelps: A tool for strain preservation and breeding programs
61(18)
Inka Bartsch
3.1 Introduction
62(1)
3.2 State of the art
63(1)
3.3 Materials
64(2)
3.3.1 Facilities and equipment for collection of sporogenous material and release of spores
64(1)
3.3.1.1 In the field
64(1)
3.3.1.2 In the laboratory
65(1)
3.3.2 Facilities and equipment list for isolation and propagation of clonal cultures
65(1)
3.3.3 Facilities and equipment list for performing laboratory-scale hybridization programs
66(1)
3.4 Experimental procedures
66(9)
3.4.1 Collection and preparation of fertile sporophytes
66(1)
3.4.2 Induced spore release
66(3)
3.4.3 Conditions to keep gametophytes vegetative
69(1)
3.4.4 Isolation of clonal gametophyte cultures
70(1)
3.4.5 Propagation of clonal cultures
71(1)
3.4.6 Hybridization of clonal gametophyte cultures
72(3)
3.5 Notes
75(1)
Acknowledgment
76(1)
References
76(3)
Chapter 4 Cryopreservation of macroalgae
79(16)
John G. Day
4.1 Introduction
79(3)
4.2 State of the art
82(4)
4.3 Materials
86(1)
4.3.1 Materials required for both methods
86(1)
4.3.2 Materials for conventional colligative cryopreservation
86(1)
4.3.3 Materials required for a vitrification-based approach
87(1)
4.4 Experimental procedures
87(3)
4.4.1 Method for conventional colligative cryopreservation
87(1)
4.4.2 Method for a vitrification-based approach
88(2)
4.5 Notes
90(1)
Acknowledgment
91(1)
References
92(3)
Chapter 5 Unraveling seaweeds bacteriomes: From field site to computer screen
95(20)
Tania Aires
Gerard Muyzer
Ester A. Serrao
Aschwin H. Engelen
5.1 Introduction
95(2)
5.2 State of the art
97(3)
5.3 Materials
100(1)
5.4 Experimental procedures
100(8)
5.4.1 Sampling
100(1)
5.4.2 DNA extraction
101(1)
5.4.3 16S rRNA amplification
102(1)
5.4.4 16S rRNA amplicon sequencing data analysis
103(2)
5.4.5 Functional prediction based on 16S rRNA gene sequencing
105(3)
5.5 Notes
108(2)
Acknowledgments
110(1)
References
110(5)
Chapter 6 Heavy metal ecotoxicity on the early life history stages of macroalgae
115(14)
Pablo P. Leal
Michael Y. Roleda
6.1 Introduction
115(1)
6.2 State of the art
116(2)
6.3 Materials
118(1)
6.3.1 Trace metal cleaning
118(1)
6.3.2 Algal material for spore release and toxicity test
118(1)
6.4 Experimental procedures
119(5)
6.4.1 Trace metal clean techniques
119(1)
6.4.2 Algal material
120(1)
6.4.3 Toxicity test
120(2)
6.4.4 End points
122(2)
6.4.5 Adaptation of the protocol to microalgae and adult macroalgal specimens
124(1)
6.5 Notes
124(1)
References
125(4)
Chapter 7 A simple protocol for a rapid and consistent production of a large number of viable protoplasts from the Ulvophycean species
129(10)
Vishal Gupta
C.R.K. Reddy
7.1 Introduction
129(1)
7.2 State of the art
130(1)
7.3 Materials
131(1)
7.3.1 Reagents
131(1)
7.3.2 Equipment
131(1)
7.3.3 Reagent setup
132(1)
7.4 Experimental procedures
132(3)
7.5 Notes
135(1)
Acknowledgments
136(1)
References
136(3)
Chapter 8 Purification of sporulation and swarming inhibitors from Ulva: Application in algal life-cycle controlling
139(20)
Ralf W. Kessler
Taghreed Alsufyani
Thomas Wichard
8.1 Introduction
140(1)
8.2 State of the art
141(1)
8.3 Materials
142(3)
8.3.1 Ulva culture
142(1)
8.3.2 Culturing of U. mutabilis on a large scale
142(1)
8.3.3 Harvesting of Ulva biomass for the extraction and the bioassay-guided fractionation of inhibitors
143(1)
8.3.4 Induction of gametogenesis/sporogenesis and bioassay-guided fractionation of the inhibitors
143(1)
8.3.5 Sporulation inhibitor 1: Extraction
144(1)
8.3.5.1 Sporulation inhibitor 1: Size-exclusion chromatography for cleanup
144(1)
8.3.6 Sporulation inhibitor 2: Extraction and bioassay-guided fractionation
144(1)
8.3.7 Swarming Inhibitor: Extraction and bioassay-guided fractionation
145(1)
8.4 Experimental procedures
145(10)
8.4.1 Cultivation of U. mutabilis under standardized conditions
145(2)
8.4.2 Induction of the gametogenesis/zoo sporogenesis and preparation of the bioassays
147(2)
8.4.3 Sporulation inhibitor 1
149(1)
8.4.3.1 Extraction of the sporulation inhibitor 1 from thallus
149(1)
8.4.3.2 Extraction of the sporulation inhibitor 1 from (axenic) growth medium
150(1)
8.4.3.3 Bioassay-guided cleanup of sporulation inhibitor 1 with size-exclusion chromatography
150(2)
8.4.4 Sporulation inhibitor 2
152(1)
8.4.4.1 Extraction of the sporulation inhibitor 2
152(1)
8.4.4.2 Bioassay-guided cleanup of the sporulation inhibitor 2
153(1)
8.4.5 Swarming inhibitor
153(1)
8.4.5.1 Extraction and bioassay-guided cleanup of the swarming inhibitor
153(1)
8.4.5.2 Bioassay-guided cleanup of the swarming inhibitor
154(1)
8.5 Notes
155(1)
Acknowledgments
156(1)
References
156(3)
Chapter 9 Preparation of axenic cultures in Ulva (Chlorophyta)
159(16)
Gianmaria Califano
Thomas Wichard
9.1 Introduction
159(2)
9.2 State of the art
161(2)
9.3 Materials
163(2)
9.3.1 Equipment
163(1)
9.3.2 Consumables and reagents
163(1)
9.3.2.1 Consumables
163(1)
9.3.2.2 PCR Reagents
164(1)
9.3.3 Ulva culture medium
164(1)
9.4 Experimental procedures
165(4)
9.4.1 Induction of gametogenesis and release of gametes
165(1)
9.4.2 Enrichment of gamete density
165(1)
9.4.3 Purification of gametes from bacteria
166(1)
9.4.4 Measuring gamete density: Flow cytometry
167(1)
9.4.5 Axenicity proof of gametes
167(1)
9.4.5.1 Test of axenicity on agar medium
168(1)
9.4.5.2 Test of axenicity by PCR
168(1)
9.5 Notes
169(1)
Acknowledgments
170(1)
References
171(4)
Section II: Chemical composition
Chapter 10 Biochar production from seaweeds
175(12)
Loretto Contreras-Porcia
Matias Araya
Elizabeth Garrido-Ramirez
Cristian Bulboa
Jean Pierre Remonsellez
Javier Zapata
Camila Espinoza
Jorge Rivas
10.1 Introduction
175(2)
10.2 State of the art
177(1)
10.3 Materials
177(3)
10.3.1 Extraction and pre-drying of seaweed biomass
177(1)
10.3.2 Sample preparation by pyrolysis
177(3)
10.3.3 Biochar generation by pyrolysis
180(1)
10.3.4 Storage of biochar samples
180(1)
10.4 Experimental procedures
180(2)
10.4.1 Extraction and pre-drying of seaweed biomass (the case of Macrocystis pyrifera)
180(1)
10.4.2 Sample preparation for pyrolysis
180(1)
10.4.3 Biochar generation by pyrolysis
181(1)
10.4.4 Storage of biochar samples
181(1)
10.5 Notes
182(1)
Acknowledgments
183(1)
References
183(4)
Chapter 11 Identification and quantification of laminarins in brown algae
187(12)
Angelika Graif
Wolfgang Ruth
Ulf Karsten
11.1 Introduction
187(2)
11.2 State of the art
189(2)
11.3 Materials
191(1)
11.3.1 Extraction of laminarin from macroalgal material
191(1)
11.3.2 Precipitation of alginate
191(1)
11.3.3 Liquid chromatography-mass spectrometrical method measurement
192(1)
11.4 Experimental procedures
192(3)
11.4.1 Laminarin extraction
192(1)
11.4.2 LC-MS measurements
193(1)
11.4.3 Laminarin reference and quantification
194(1)
11.5 Notes
195(1)
Acknowledgment
196(1)
References
197(2)
Chapter 12 Determination of carbohydrate composition of macroalgae
199(12)
Wouter J.J. Huijgen
E.M. Cobussen-Pool
B.F. van Egmond
J.W. van Hal
12.1 Introduction
200(1)
12.2 State of the art
201(1)
12.3 Materials
202(1)
12.3.1 Seaweed
202(1)
12.3.2 Reagents
202(1)
12.3.3 Equipment
203(1)
12.4 Experimental procedures
203(5)
12.4.1 Preparation seaweed sample
204(1)
12.4.2 Hydrolysis
204(1)
12.4.2.1 Prehydrolysis
204(1)
12.4.2.2 Hydrolysis
205(1)
12.4.3 Analysis monomeric carbohydrates
205(1)
12.4.3.1 Sample preparation
205(1)
12.4.3.2 Gradient
206(2)
12.4.4 Calculations
208(1)
12.5 Notes
208(2)
12.5.1 General remarks
208(1)
12.5.2 Seaweed hydrolysis
209(1)
12.5.3 High-performance anion-exchange chromatography with pulsed amperometric detector
209(1)
Acknowledgments
210(1)
References
210(1)
Chapter 13 Quantification of proteins in seaweeds
211(14)
Carl Safi
Jelle van Leeuwen
Yvette Telleman
Nicole Engelen-Smit
Lambertus van den Broek
Paulien Harmsen
13.1 Introduction
212(1)
13.2 State of the art
212(1)
13.3 Materials
213(4)
13.3.1 Kjeldahl method: Total protein determination
213(1)
13.3.2 Dumas method: Total protein determination
213(3)
13.3.3 Amino acid determination
216(1)
13.3.4 Nitrogen-to-protein-conversion factor determination
216(1)
13.3.5 Lowry method: Soluble protein determination
216(1)
13.3.6 Bradford method: Soluble protein assay
217(1)
13.4 Experimental procedures
217(5)
13.4.1 Kjeldahl method: Total protein determination
217(1)
13.4.1.1 Digestion
217(1)
13.4.1.2 Distillation
217(1)
13.4.1.3 Titration
218(1)
13.4.1.4 Calculation
218(1)
13.4.2 Dumas method: Total protein determination
218(1)
13.4.2.1 Procedure
218(1)
13.4.3 Amino acid determination
219(1)
13.4.3.1 Preparation of reagents
219(1)
13.4.3.2 Preparation of eluents
219(1)
13.4.3.3 Preparation of standards
219(1)
13.4.3.4 Acid hydrolysis
220(1)
13.4.3.5 Analysis
220(1)
13.4.4 Nitrogen-to-protein-conversion factor determination
220(1)
13.4.4.1 Procedure
220(1)
13.4.5 Lowry method: Soluble protein determination
221(1)
13.4.5.1 Preparation of the standard solution
221(1)
13.4.5.2 Preparation of Folin-Ciocalteau reagent
221(1)
13.4.5.3 Test tube protocol
222(1)
13.4.6 Bradford method: Soluble protein assay
222(1)
13.4.6.1 Procedure
222(1)
13.5 Notes
222(1)
Acknowledgment
223(1)
References
223(2)
Chapter 14 Comprehensive phytohormone quantification in the red alga Pyropia yezoensis by liquid chromatography-mass spectrometry
225(12)
Takakazu Matsuura
Izumi C. Mori
Yoko Ikeda
Takashi Hirayama
Koji Mikami
14.1 Introduction
226(1)
14.2 State of the art
226(1)
14.3 Materials
227(1)
14.3.1 Extraction and purification of phytohormones
227(1)
14.3.2 Liquid chromatography-mass spectrometry
228(1)
14.4 Experimental procedures
228(6)
14.4.1 Extraction
229(1)
14.4.2 Partial purification of phytohormones with solid-phase extraction
229(2)
14.4.3 Liquid chromatography-mass spectrometry analysis
231(3)
14.4.4 Data analysis
234(1)
14.5 Notes
234(2)
References
236(1)
Chapter 15 Total phenolic content and antioxidant capacity analysis of seaweed biomass
237(12)
Xiaoru Hou
Randi Neerup
Anne-Belinda Bjerre
15.1 Introduction
237(1)
15.2 State of the art
238(1)
15.3 Materials
239(2)
15.3.1 Seaweed biomass
239(1)
15.3.2 Equipment
240(1)
15.3.3 Chemicals
240(1)
15.3.3.1 Total phenolic content analysis of seaweed biomass
240(1)
15.3.3.2 Antioxidant capacity analysis of seaweed biomass: 2,2-Diphenyl-1-picrylhydrazyl assay
241(1)
15.4 Experimental procedures
241(6)
15.4.1 Total phenolic content analysis of seaweed biomass
241(1)
15.4.1.1 Folin-Ciocalteu assay
241(1)
15.4.1.2 Prussian blue assay
243(2)
15.4.2 Antioxidant capacity analysis of seaweed biomass: 2,2-Diphenyl-1-picrylhydrazyl assay
245(2)
15.5 Tips and tricks
247(1)
Acknowledgment
247(1)
References
247(2)
Chapter 16 Extraction of phycocyanin and phycoerythrin pigments
249(18)
Stewart William Beattie
Michele Morancais
Paul Delens
Joel Fleurence
Justine Dumay
16.1 Introduction
250(1)
16.2 State of the art
251(1)
16.3 Materials
252(2)
16.3.1 Algal harvesting
252(1)
16.3.2 Algal conditioning
253(1)
16.3.3 Buffer preparation
254(1)
16.4 Experimental procedures
254(5)
16.4.1 Phycobiliproteins extraction
254(1)
16.4.1.1 Algal treatment
254(1)
16.4.1.2 Maceration
254(1)
16.4.1.3 Acid extraction
255(1)
16.4.1.4 Ultrasonic treatment
255(1)
16.4.1.5 Enzymatic hydrolysis
255(1)
16.4.2 Phycobiliprotein purification
256(1)
16.4.2.1 Adsorption bed chromatography
256(1)
16.4.2.2 Ammonium sulphate precipitation
257(1)
16.4.2.3 Ion-exchange column
257(1)
16.4.2.4 Hydroxyapatite chromatography
257(1)
16.4.2.5 Aqueous two-phase extraction
258(1)
16.4.3 Analytical method
258(1)
16.5 Concluding remarks
259(1)
16.6 Notes
260(2)
References
262(5)
Chapter 17 Quantification and localization of reactive oxygen species in marine macrophytes
267(12)
Manoj Kumar
Loretto Contreras-Porcia
Nirali M. Kumar
Peter J. Ralph
17.1 Introduction
268(1)
17.2 State of the art
269(4)
17.2.1 ROS detection reaction mechanism using dihydroethidium and dichlorodihydrofluorescein diacetate
269(2)
17.2.2 Histochemical localization mechanism using nitro blue tetrazolium and DAB staining
271(2)
17.3 Material
273(1)
17.3.1 Reactive oxygen species visualization by microscopy
273(1)
17.3.2 Reactive oxygen species quantification using dihydroethidium and dichlorodihydrofluorescein diacetate
273(1)
17.3.3 ROS histochemical localization
273(1)
17.4 Experimental procedures
274(1)
17.4.1 Reactive oxygen species visualization by microscopy
274(1)
17.4.2 ROS quantification using DHE and DCFH-DA
274(1)
17.4.3 Histochemical localization for reactive oxygen species detection
274(1)
17.5 Tips and tricks
275(1)
17.5.1 Reactive oxygen species visualization
275(1)
17.5.2 Reactive oxygen species quantification
275(1)
17.5.3 Reactive oxygen species histochemical localization
276(1)
Acknowledgment
276(1)
References
276(3)
Chapter 18 Metabolomics of intra- and extracellular metabolites from micro- and macroalgae using GC-MS and LC-MS
279(22)
Constanze Kuhlisch
Gianmaria Califano
Thomas Wichard
Georg Pohnert
18.1 Introduction
280(1)
18.2 State of the art
281(2)
18.3 Materials
283(4)
18.3.1 Solvents
283(1)
18.3.2 Equipment
283(2)
18.3.3 Consumables
285(1)
18.3.4 Solution recipes
285(1)
18.3.5 Instrumental setup
286(1)
18.4 Experimental procedures
287(8)
18.4.1 Sampling and metabolic quenching
288(1)
18.4.1.1 Filtration of planktonic single-celled algae
288(1)
18.4.1.2 Collection of algal gametes (of Ulva spp.)
288(1)
18.4.1.3 Collection of algal thalli
289(1)
18.4.2 Solid-phase extraction of extracellular metabolites
289(1)
18.4.3 Extraction of intracellular metabolites
290(1)
18.4.3.1 Cell disruption by ultrasound treatment
290(1)
18.4.3.2 Cell disruption with a bead mill
290(1)
18.4.4 Two-step-derivatization for GC-MS analysis
291(1)
18.4.5 GC-MS analysis
292(1)
18.4.6 Data analysis for GC-MS data
292(3)
18.5 Notes
295(2)
Acknowledgments
297(1)
References
297(4)
Chapter 19 Preparative extraction of exometabolites from seaweed surfaces
301(8)
Florian Weinberger
19.1 Introduction
301(1)
19.2 State of the art
302(2)
19.3 Materials
304(1)
19.4 Experimental procedures
304(3)
19.4.1 Development of extraction protocol
304(1)
19.4.2 Determination of algal surface/mass ratio
305(1)
19.4.3 Extraction
306(1)
19.5 Notes
307(1)
References
307(2)
Chapter 20 Disruption-free solid-phase extraction of surface metabolites from macroalgae
309(14)
Emilio Cirri
Georg Pohnert
20.1 Introduction
309(1)
20.2 State of the art
310(1)
20.3 Materials
311(1)
20.3.1 Fucus vesiculosus
311(1)
20.3.2 Solid-phase surface extraction method
312(1)
20.4 Experimental procedures
312(3)
20.4.1 Extraction procedure
313(2)
20.4.2 Data evaluation
315(1)
20.5 Notes
315(2)
Acknowledgments
317(1)
References
317(6)
Section III: Cellular and molecular characterization
Chapter 21 The immunodetection and in situ imaging of cell-wall polysaccharides in brown algae
323(12)
Amandine Simeon
Delphine Duffieux
Cecile Herve
Sophie Le Panse
Paul Knox
Thomas Torode
21.1 Introduction
323(1)
21.2 State of the art: Imaging cell walls in brown algae
324(1)
21.3 Materials
325(2)
21.4 Experimental procedures
327(5)
21.4.1 Preparation of algal material and fixation procedures
327(1)
21.4.2 Embedding protocol for London resins White resin
328(1)
21.4.3 Embedding protocol for Lowicryl resin
329(1)
21.4.4 Sectioning of resin-embedded samples
330(1)
21.4.5 Imaging algal cell walls using general stains
330(1)
21.4.6 Immunolabeling for fluorescence microscopy
330(1)
21.4.7 Immunolabeling for electron microscopy
331(1)
21.4.8 Pretreatments before immunolabeling
331(1)
21.5 Notes
332(1)
Acknowledgments
333(1)
References
333(2)
Chapter 22 Atomic force microscopy based analysis of cell-wall elasticity in macroalgae
335(14)
Thomas Torode
Marina Linardic
J. Louis Kaplan
Siobhan A. Braybrook
22.1 Introduction
335(1)
22.1.1 Biomechanical characterization of macroalgal cell walls
335(1)
22.2 State of the art
336(2)
22.2.1 Mechanical characterization of macroalgal cell walls
336(2)
22.3 Materials
338(1)
22.4 Experimental procedures
338(7)
22.4.1 Preparation of algal material for atomic force microscopy analysis
338(1)
22.4.2 Choosing a force for indentation
339(2)
22.4.3 Influence of osmoticum upon macroalgal mechanics
341(1)
22.4.4 Selection of tip size
342(1)
22.4.5 Performing atomic force microscopy based elasticity measurements
343(2)
22.5 Notes
345(1)
Acknowledgments
346(1)
References
346(3)
Chapter 23 Dynamic and microscale mapping of cell growth: Case of Ectocarpus filament cells
349(16)
Herve Rabille
Bernard Billoud
Elodie Rolland
Benedicte Charrier
23.1 Introduction
350(1)
23.2 State of the art
351(2)
23.3 Materials
353(1)
23.3.1 Reagents
353(1)
23.3.2 Ectocarpus culture
353(1)
23.3.3 Equipment and software
353(1)
23.4 Experimental procedures
354(6)
23.4.1 Preparation of living algal material for time-lapse microscopy
354(1)
23.4.1.1 Gametes release on glass-bottom Petri dish, multiwell plate or free coverslips
354(1)
23.4.1.2 Preparation of "homemade" glass bottom Petri dishes
354(1)
23.4.2 Cell-surface deformation monitoring using time-lapse microscopy
355(1)
23.4.2.1 Prior preparation of the equipment
356(1)
23.4.2.2 Cell-surface labeling with fluorescent microspheres
356(1)
23.4.2.3 Measuring irreversible deformations during growth (plastic strain)
356(1)
23.4.2.4 Measuring deformations induced by hypo- or hypertonic shock (mainly elastic deformations)
357(2)
23.4.3 Picture analysis: Quantification of cell-surface marker displacement
359(1)
23.5 Notes
360(2)
References
362(3)
Chapter 24 Actin fluorescent staining in the filamentous brown alga Ectocarpus siliculosus
365(16)
Herve Rabille
Maria Koutalianou
Benedicte Charrier
Christos Katsaros
24.1 Introduction
365(2)
24.2 State of the art
367(2)
24.3 Materials
369(4)
24.3.1 Chemicals
369(1)
24.3.2 Solutions and recipes
369(1)
24.3.2.1 Preparation of Rhodamine-Phalloidin (R415) or AlexaFluor568-Phalloidin (A12380, life technology)
369(1)
24.3.2.2 Preparation of buffers
370(1)
24.3.2.3 Other solutions
372(1)
24.3.3 Equipment
373(1)
24.4 Experimental procedures
373(2)
24.4.1 Preparation of poly-L-lysine-coated coverslips
373(1)
24.4.2 Preparation of Ectocarpus samples
373(1)
24.4.2.1 Filaments grown on coverslips
373(1)
24.4.2.2 Free-floating filaments
374(1)
24.4.3 Actin staining procedure
374(1)
24.5 Notes
375(1)
Acknowledgments
376(1)
References
376(5)
Chapter 25 Cryofixation of brown algae for transmission electron microscopy
381(10)
Chikako Nagasato
Christos Katsaros
Taizo Motomura
25.1 Introduction
381(2)
25.2 State of the art
383(1)
25.3 Materials
384(1)
25.3.1 Reagents
384(1)
25.3.2 Equipment
384(1)
25.4 Experimental procedures
384(3)
25.4.1 Preparation of material
384(1)
25.4.2 Cryogen and substitution fixative
385(1)
25.4.3 Cryofixation
386(1)
25.4.4 Freeze substitution
386(1)
25.4.5 Infiltration and embedding of samples into resin
386(1)
25.5 Notes
387(1)
25.6 Concluding remarks
387(1)
References
388(3)
Chapter 26 Probing the subcellular topography of seaweeds: Transmission electron microscopy, immunocytochemistry, and correlative light microscopy
391(20)
Sandra C. Raimundo
David S. Domozych
26.1 Introduction
392(1)
26.2 State of the art
393(2)
26.2.1 General considerations
393(1)
26.2.2 Protocol considerations
394(1)
26.3 Materials
395(4)
26.3.1 Fixation, dehydration, and embedding
395(2)
26.3.2 Polymerization
397(1)
26.3.3 Sectioning
397(1)
26.3.4 Uranyl acetate/Lead citrate staining
397(1)
26.3.5 Immunogold labeling
398(1)
26.4 Experimental procedures
399(8)
26.4.1 Fixation
399(1)
26.4.2 Dehydration
399(1)
26.4.3 Infiltration
400(1)
26.4.4 Polymerization
400(1)
26.4.5 Sectioning
401(2)
26.4.6 Uranyl acetate/Lead citrate staining
403(1)
26.4.7 Immunogold labeling
404(1)
26.4.8 Considerations for light microscopy and correlative studies
405(2)
26.5 Notes
407(2)
References
409(2)
Chapter 27 Coralline algae preparation for scanning electron microscopy and optical microscopy
411(18)
S. Kaleb
G. Alongi
A. Falace
27.1 Introduction
411(1)
27.2 State of the art
412(1)
27.3 Materials
412(2)
27.3.1 Materials for optical microscopy
412(1)
27.3.2 Materials for scanning electron microscopy
413(1)
27.4 Experimental procedures
414(11)
27.4.1 Choice of the portion to cut and orientation of the sample
414(6)
27.4.2 Optical microscopy
420(3)
27.4.3 Scanning electron microscopy
423(2)
27.5 Notes
425(2)
Acknowledgments
427(1)
References
427(2)
Chapter 28 Extraction of high quality RNA from brown algae for transcriptomic analysis
429(12)
Sandra Heinrich
28.1 Introduction
429(1)
28.2 State of the art
430(2)
28.3 Materials
432(1)
28.3.1 Algal material
432(1)
28.3.2 Sample homogenization
432(1)
28.3.3 RNA extraction
432(1)
28.3.3.1 Reagents
432(1)
28.3.3.2 Equipment
432(1)
28.3.4 RNA quality control
433(1)
28.4 Experimental procedures
433(3)
28.4.1 Sample homogenization
433(1)
28.4.2 RNA extraction
433(2)
28.4.3 RNA quality control
435(1)
28.4.3.1 Measuring RNA concentration and purity
435(1)
28.4.3.2 RNA integrity control
436(1)
28.5 Notes
436(1)
Acknowledgments
437(1)
References
437(4)
Chapter 29 Induction of sexual reproduction in Spirogyra cultures for laser capture microdissection of gametes and zygotes
441(12)
Denis Saint-Marcoux
Jane A. Langdale
29.1 Introduction
441(2)
29.2 State of the art
443(1)
29.3 Materials
444(1)
29.3.1 Equipment
444(1)
29.3.1.1 Algae cultivation
444(1)
29.3.1.2 Laser microdissection
444(1)
29.3.2 Reagents
445(1)
29.4 Experimental procedures
445(4)
29.4.1 Cultivation of Spirogyra and induction of sexual reproduction
445(1)
29.4.1.1 Media preparation
445(1)
29.4.1.2 Vegetative growth
446(1)
29.4.1.3 Sexual reproduction induction
446(1)
29.4.2 Fixation of thallus before laser capture microdissection
447(2)
29.4.3 Laser capture microdissection
449(1)
29.5 Notes
449(2)
Acknowledgment
451(1)
References
451(2)
Chapter 30 Cloning and expression strategies for the postgenomic analysis of brown algae
453(16)
Agnes Groisillier
30.1 Introduction
454(1)
30.2 State of the art
455(1)
30.3 Materials
456(3)
30.3.1 Strains and vector
457(1)
30.3.1.1 E. coli
457(1)
30.3.1.2 Vector
457(1)
30.3.2 General stock solutions
457(1)
30.3.3 PCR, cloning and transformation procedures
457(1)
30.3.4 Protein expression and purification
458(1)
30.3.5 Equipment
458(1)
30.3.6 Software
459(1)
30.4 Experimental procedures
459(5)
30.4.1 Bioinformatics analysis
459(1)
30.4.2 Cloning
459(1)
30.4.2.1 Amplification of gene of interest
459(1)
30.4.2.2 PCR fragment purification
460(1)
30.4.2.3 PCR fragment digestion
460(1)
30.4.2.4 pFO4 vector preparation
460(1)
30.4.2.5 Ligation step
461(1)
30.4.2.6 Transformation in E. coli DH5a
461(1)
30.4.2.7 PCR screening
461(1)
30.4.2.8 Plasmid extraction and glycerol stock
462(1)
30.4.2.9 Transformation of expression strain, PCR screening, and glycerol stock
462(1)
30.4.3 Small-scale test expression
462(1)
30.4.3.1 Cultivation was performed in two phases
462(1)
30.4.3.2 Lysis of cells and detection of proteins
462(1)
30.4.4 Large-scale expression and purification
463(1)
30.4.4.1 Expression culture
463(1)
30.4.4.2 Affinity chromatography
463(1)
30.4.4.3 Size-exclusion chromatography
463(1)
30.4.4.4 Analysis of the chromatography
463(1)
30.5 Notes
464(2)
References
466(3)
Chapter 31 Polyethylene glycol-mediated transformation in the green macroalga Ulva mutabilis (Chlorophyta): A forward genetics approach
469(16)
Jens Boesger
Michiel Kwantes
Thomas Wichard
31.1 Introduction
470(1)
31.2 State of the art
471(3)
31.3 Materials.,
474(1)
31.3.1 Equipment, consumables, and reagents
474(1)
31.3.1.1 Equipment and consumables
474(1)
31.3.1.2 Reagents
474(1)
31.3.2 Ulva culture medium
475(1)
31.4 Experimental procedures
475(6)
31.4.1 Appropriate vector preparation for transformation of U. mutabilis
475(1)
31.4.2 Preparation of gametes of U. mutabilis for transformation
475(1)
31.4.3 Transformation of U. mutabilis gametes
476(1)
31.4.4 Cultivation of transgenic Ulva lines and selection by phleomycin treatment
477(1)
31.4.5 Determination of the transformation efficiency and further single-strain cultivation
478(1)
31.4.6 Confirmation of stable transformation
478(3)
31.5 Notes
481(1)
Acknowledgments
482(1)
References
482(3)
Index 485
Dr. Bénédicte Charrier is a senior scientist from CNRS working at Station Biologique Roscoff, France. She is a specialist of macroalgae morphogenesis and development, with an expertise on the filmentous alga Ectocarpus siliculosus. She is currently the Chair of the European network "Phycomorph", supported by the Cooperation in Science & Technology (COST) Association (2015-2019), which aims to coordinate research in growth, reproduction, and morphogenesis of macroalgae in Europe and in association with Asian and North American laboratories.



Dr. Thomas Wichard is a research group leader at the Institute forInorganic and Analytical Chemistry of the Friedrich SchillerUniversity Jena. The main focus of his research group is to elucidatethe mutualistic interactions between bacteria and the marine macroalgaUlva ("cross-kingdom-cross-talk"). The group applies variousmethodologies in analytical chemistry, chemical ecology and molecularbiology to understand the basis of eco-physiological processes.



Dr. CRK Reddy is a Chief Scientist in Seaweed Biology and Biotechnology Cultivation at CSIR-Central Salt and Marine Chemicals Research Institute in Bhavnagar, India. He is an expert in marine macroalgae, making significant contributions to macroalgae tissue culture and protoplast techniques including genetic improvement of seaweeds, seaweed biomass for chemicals and biofuels, seaweed biodiversity, and seaweed biology and cultivation.