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Defence on surface of Rhodophyta Halymenia floresii :
metabolomic profile and interactions with its
surface-associated bacteria
Shareen a Abdul Malik
To cite this version:
Shareen a Abdul Malik. Defence on surface of Rhodophyta Halymenia floresii : metabolomic profile and interactions with its surface-associated bacteria. Agricultural sciences. Université de Bretagne Sud, 2020. English. �NNT : 2020LORIS598�. �tel-03153270�
Par
Shareen A ABDUL MALIK
T
HESE DE DOCTORAT DE
UNIVERSITE
BRETAGNE
SUD
ECOLE DOCTORALE N°598
Sciences de la Mer et du littoral
Spécialité : Biotechnologie Marine
Defence on surface of Rhodophyta Halymenia floresii: metabolomic
fingerprint and interactions with the surface-associated bacteria
Thèse présentée et soutenue à « Vannes », le « 7 July 2020 »
Unité de recherche : Laboratoire de Biotechnologie et Chimie Marines
Thèse N°:
Rapporteurs avant soutenance :
Prof. Gérald Culioli Associate Professor
Université de Toulon (La Garde)
Dr. Leila Tirichine Research Director (CNRS)
Université de Nantes
Composition du Jury :
Président :
Prof. Claire Gachon Professor
Museum National d’Histoire Naturelle, Paris
Examinateur(s) :
Prof. Gwenaëlle Le Blay Professor
Université Bretagne Occidentale (UBO), Brest
Dir. de thèse :
Prof. Nathalie Bourgougnon Professor
Université Bretagne Sud (UBS), Vannes Co-dir. de thèse :
Title: Systèmes de défence de surface de la Rhodophycée Halymenia floresii : Analyse metabolomique et interactions avec les bactéries épiphytes
Mots clés: Halymenia floresii, antibiofilm, antifouling, métabolomique, bactéries associées à la surface, quorum sensing, molecules de défense
Title: Defence on surface of Rhodophyta Halymenia floresii: metabolomic fingerprint and interactions with the surface-associated bacteria
Keywords: Halymenia floresii, antibiofilm, antifouling, metabolomics, surface-associated bacteria, quorum
sensing, defence molecules
Abstract : Halymenia floresii, une Rhodophycée présente une surface remarquablement exempte d'épiphytes dans les conditions de l'Aquaculture MultiTrophique Intégrée (AMTI). Ce phénomène la présence en surface de composés actifs allélopathiques. L'objectif de ce travail a été d'explorer les mécanismes de défense développés par H. floresii contre l'épibiose, de détecter et d'identifier les métabolites secondaires produits à la surface de l’algue et d'étudier les relations avec les bactéries épiphytes. Nous avons ainsi pu isoler la communauté épibactérienne de H. floresii cultivée dans des conditions contrôlées (AMTI) et non contrôlées (échantillons collectés in situ). Les épibactéries isolées ont été criblées in vitro pour analyser les signaux de détection de Quorum Sensing (QS). Lesextraits produits en surface ont été analysés pour détecter toute interférence avec le Quorum Sensing. Les épibactéries pathogènes et
non-pathogènes ont été différenciées par leur capacité à induire
une maladie algale, le blanchiment.
Vibrio owensii, ainsi que son signal C4-HSL QS, a été
identifié comme pathogène opportuniste induisant un blanchiment. Les métabolites extraits de la surface et de cellules entières de H. floresii ont été analysés par LC-MS. Une base de données a été constituée à partir d’une analyse métabolomique non ciblée. Quarante et un putatives actifs ont été identifiés, parmi lesquels les composés halogénés, des furanones et divers inhibiteurs étaient surreprésentés. Fait intéressant, les deux premières classes sont connues comme de puissants composés interférant avec le QS. La
présence relativement élevée de putatives
allélopathiques à la surface de H. floresii soutient fortement l'hypothèse selon laquelle ils doivent être impliqués dans la protection de l'hôte. Des recherches supplémentaires seront nécessaires pour explorer l’ensemble des métabolites secondaires produits par
H. floresii et leurs rôles chez l’algue.
Abstract: The surface of Halymenia floresii, a Mexican
Rhodophyta, was observed to be remarkably free of epiphytes under Integrated MultiTrophic Aquaculture (IMTA) conditions. This suggests the presence of allelopathic active compounds released by this macroalgae. The aim of this work was to explore the defence mechanisms developed by H. floresii against surface epibiosis, to detect and identify the secondary metabolites produced at the surface of the alga, and to study its relation with surface associated bacteria. For the first time, we isolated the epibacterial community of H.
floresii cultivated under controlled conditions (IMTA) and
uncontrolled ones (beach-cast material collected in the area). The isolated epibacteria were screened in vitro to analyse Quorum Sensing (QS) signals, and others H.
floresii surface extracts were assayed for any QS
interference with them. We differentiated the epibacteria significant pathogens from the non-pathogens ones by their ability to induce bleaching, a well-known algal disease.
Vibrio owensii was identified as an opportunistic
pathogen inducing bleaching in H. floresii which was also associated to the presence of its C4-HSL QS signal. The surface and whole cell metabolites extracts from H. floresii specimens cultivated under controlled conditions were analysed by means of LC/MS. An untargeted metabolomic analysis of H.
floresii was performed to provide a global metabolic
fingerprint as a first baseline. We identified ‘41’ active putatives in H. floresii, among which halogenated compounds, furanones and various inhibitors were overrepresented. Interestingly, the first two classes are well known potent QS interfering compounds. The relatively higher occurrences of allelopathic putatives at the surface of H. floresii strongly supports the hypothesis that they must be involved in the host protection. Further investigations are needed to explore the secondary metabolites of H.
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cup, it becomes the cup; You put water into a bottle it becomes the bottle; You put
it in a teapot it becomes the teapot. Now water can flow or it can crash. Be water,
my friend.
- Bruce Lee
Be careful what you water your dreams with. Water them with worry and fear
and you will produce weeds that choke the life from your dream. Water them with
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Acknowledgements
Wow... Expressing gratitude is such a greatest human emotion and it’s very exciting to start acknowledging the BEings who have been and/or being with me throughout my PhD. This period of ‘3’ years in my life gave me a lot of people and lots and lots of wonderful experiences. And I am very grateful for it.
Firstly, I sincerely thank Prof. Gérald Culioli, Associate Professor of the Laboratory of Matériaux Polymères Interfaces Environnement Marin (MAPIEM), Université de Toulon (La Garde) and Dr. Leila Tirichine, Research Director of the Epigénomique des microalgues et interactions avec l’environment, Université de Nantes, for being rapporteurs and examining the work. I also thank other members of jury Prof. Claire Gachon, Professor, Museum National d’Histoire Naturelle and Prof. Gwenaëlle Le Blay, Professor, Université Bretagne Occidentale (UBO) for equally participating in examining my work.
I profoundly thank my doctoral school, Ecole Doctorale Sciences de la Mer et du Littoral (EDSML) for constantly supporting throughout my thesis and granting me scholarship for my international mobility. I also thank ECOS-Nord CONACYT for their financial support towards my research stay in Mexico. I sincerely thank GlobalSeaweedSTAR for their financial help towards attending the conference of ISS, 2019, Korea.
Well, my Directors of the thesis... Words are not enough to express my deep gratitude to Prof. Nathalie Bourgougnon, firstly for the offering me this wonderful opportunity to pursue my PhD under her wonderful supervision. Her immense knowledge always influenced me to think critically and develop my ideas. I always admired her way of valuing others TIME equally as she values her own. I could not have imagined having a better supervisor for my PhD study. I equally thank my Co-director Dr. Daniel Robledo for sharing his knowledge on his all-time favourite, macroalgae. I really appreciate his guidance in writing the publications for his insightful comments which incented me to widen my perspectives of the subject. Nothing would have been possible without their continuous support to my PhD.
My sincere thanks also go to Dr. Gilles Bedoux for his valuable comments and guidance in the chemistry part of the research and his advice on chromatographic analyses is highly valuable. I greatly appreciate his deep knowledge on the subject which really ignited my interest in the field of chemistry. I extend my gratitude to Dr. José Q. Garcia Maldonado for his guidance and comments in the molecular biology part of the research. His direction really helped me a lot and earned me a confidence in the field of molecular biology. I also gratefully acknowledge Prof. Alain Dufour and Dr. Alexis Bazire for their guidance and views in the quorum sensing related work of the research. I also sincerely thank Dr. Bazire for offering me
the reporter strains that really helped me carry out the QS related works. I also deeply thank Dr. Yolanda Freile-Pelegrín for her care and concern during my research stay in Mexico and also for the GC-MS instrument in her lab for analysing my extracts. Furthermore, I extend my deepest gratitude to Dr. Mahasweta Saha (Plymouth Marine Laboratory, UK) for her very detailed description and guidance through the disease part of the work. I also thank Mme. Florence Le Sann, Mme. Noluenn Chauvin and Mme. Axelle Guitton for their administrative help accompanying throughout the thesis, regarding the mobility’s and conferences. I also thank Yohan Duhautois for his administrative assistance regarding the formations.
Technicians are the real heroes of any lab. As my research includes different laboratories, I had an opportunity to meet many heroes during my thesis. First of all, Christel Marty (LBCM, France), I truly thank her for teaching and explaining me the preventive measures of the laboratory and being very supportive all through my research in LBCM. My heartfelt thanks to Crescencia and Edgar Caamal-Fuentes (Phycology, Mexico) for all their support and help, both professionally and morally throughout my stay in Mexico. I will never forget Cres’s help in finding me accommodation for my relaxed stay in Merida. Edgar’s knowledge on chemistry, particularly on GC-MS, always astonished me and I am very grateful for sharing it with me. I also thank V. Avila-Velazquez for his skillful technical assistance for the algal cultivation.
My earnest thanks to Philippe Douzenel (UBS, France) for his great help and support in carrying out different experimentations and especially for his support in HPLC related works. My heartfelt thanks extend to Laure Taupin (LBCM, France) for her tremendous and wonderful help and support in LC-MS related works. I should definitely mention her fastest reply to my doubts and her availability in sending me the results. I wholeheartedly thank Bernard Lassard (UBS, France) for all his help and support and very specially for the Luminometer. Being a very happy person, Bernard always gave me answers for my questions and never judged me for my silly doubts. And Abril Gamboa and Roscita (Parasitology and Molecular Biology, Mexico), truly I thank both of them for their immense support in molecular works and maintaining my bacteria. I deeply appreciate their efforts in understanding my needs though I could able to communicate only in English with them. I also truly thank Ana and Dora (Microscopy, Mexico) for their patience and assistance in travelling with me over the surface of my alga and finally getting good pictures of the microbiome.
And my team….LBCM… I should obviously start with Maya with whom I started my PhD days. Thank you, Maya for teaching and assisting me to learn basic experimentations in my early days. I sincerely thank Anne-Sophie getting me the algal samples from Mexico. I wholeheartedly thank Florence for BEing with
me during the very first isolation of my bacteria. I am also happy that I had a chance to meet Romain who also helped me learning basic manipulations. I happily thank Nolwenn, Marie and Kevin for having been there for me and I shall definitely mention about my coffee time with Nolwenn and chatting about the whole world. I have only few people in UBS to talk with and Hugo, you are first among them. Thank you very much, my friend for everything! For sharing your experiences and stories and moreover your family with me. I think of Paola, Emiliano, Rebecca at this time and of course, Gael. And IRDL… I sincerely owe my gratitude to the whole team for the beautiful smiling morning wishes.
And my team…. Phycology…. I dearly thank Ana for the beautiful friendship and all those big laughs we shared and of course Adrian too, though he did not have a clue of what we were talking he laughed with us. Well…now, Parasitology, I thank each and every one of the team for the wonderful everlasting experiences, in and out of the lab! I sincerely extend my gratitude to Dr. Victor and Dr. Leo for their very warm love and affection and especially for the Piñata during Christmas! I also thank them for allowing me to use the Epifluorescence microscope. I am very grateful to the whole team. Thanking the friends is not my way, I extend all my love and care to Karen, Eric, Roman, Fer, Sonia, Regina, Celina, Danilu, Catherine and I promise to stand by them as far as I can. I thank Danilu and Catherine, together with their family, for giving me most memorable days in my life by incorporating me in their life.
I also thank Mr. Jean-Roch Sauvé for the pleasant ride to Lorient for few of my manipulations there. Your help really matters a lot for me and moreover I cannot forget the joke you prepared for your daughter on 1st
of April. And finally, Olivier Sire… Thank you for everything! I deeply extend my gratitude and love for BEing with me through my hard times, Just BE, my friend. Thank you very much, Olivier, particularly for clearing the fog on my way and walking me through it.
And my Family… it is hard to find words to thank them. Thank you Mummy…Machi…Thashu…Sunil ma…Kuttima…Mithu… and my dear Don. Thank you, Mummy for believing in me even at times when even I didn’t believe me. I promise you all that my every single action will make you all proud. And Raphael… Thank you da for being such a wonderful companion, for being my soul mate, roommate and my everything. Love you all forever and ever.
I should definitely mention few of several invisible people and friends who were with me in this journey. I wholeheartedly thank Ambika, Navashree annae and Sahana who supported me during all my visas processes. And I also thank Vimal annae, Baba annae, Sangeeth annae and all friends in Divonne-les bains who helped us commencing our journey to Bretagne and for all their moral support.
And To My DADDY… I made of one of your several DREAMS for me to come alive now…! Thank you for protecting me from Ignorance and enlightening me with the Wisdom, Daddy! JUST BE, Daddy! LOVE YOU FOREVER!
List of Publications
The work of this thesis was published by means of the following publications.
Chapter I
A Abdul Malik S., Bedoux G., Garcia Maldonado J. Q., Freile-Pelegrín Y., Robledo D., & Bourgougnon N. (2020) Chapter 10. Defense on surface: macroalgae and their surface-associated microbiome. Seaweeds Around the World: State of Art and Perspectives. Advances Botanical research, Volume 95. ISBN: 978-0-08-102710-3.
Chapter II
A Abdul Malik S., Bazire A., Gamboa A., Bedoux G., Robledo D., Garcia Maldonado J. Q., & Bourgougnon N. (2020) Quorum Sensing screening in the surface-associated bacteria of the Mexican red
alga, Halymenia floresii. Microbiology, accepted (In press) (Manuscript ID:
MICBIO2004A005AABDULM) Chapter III
A Abdul Malik S., Bedoux G., Robledo D., Garcia Maldonado J. Q., Freile-Pelegrín Y., & Bourgougnon N. (2020) Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta). Journal of Applied Phycology 10.1007/s10811-020-02094-4.
Chapter IV
A Abdul Malik S., Saha M., Bedoux G., Robledo D., & Bourgougnon N. Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method (under preparation).
Chapter V
A Abdul Malik S., Bedoux G., Freile-Pelegrín Y., Bourgougnon N., & Robledo D. (2020) Screening
Halymenia floresii secondary metabolites which underly its surface defence mechanisms. Metabolites, submitted (Manuscript ID: metabolites-956819).
Table of Contents
1. General Introduction ...
2
1.1 Part I – Introduction to Macroalgae ...
2
1.1.1 Macroalgae ...
2
1.1.2 History and Classification ...
2
1.1.3 Ecology ...
3
1.1.4 Valorization of Macroalgae ...
4
1.1.5 Macroalgae in Mexico ...
6
1.2 Part – II– Red Macroalgae ...
8
1.2.1 Introduction ...
8
1.2.2 Biology of Red Macroalgae ...
10
i. Cell wall ...
10
ii. Polysaccharides: Structural and Storage ...
11
a. Carrageenans ...
11
b. Agar ...
13
c.
Starch ...13
iii. Pigments and Proteins ...
16
iv. Lipids ...
18
1.2.3 Species under Study - Halymenia floresii ...
18
1.3 Part – III- Abiotic and Biotic parameters ...
23
1.3.1 Introduction ...
23
1.3.2 Definition and Types - Abiotic parameters ...
24
i. Hydrodynamism ...
24
ii. Nutrients ...25
iii. Desiccation ...25
iv. Temperature ...26
v. Light ...26
vi. Salinity ...26
1.3.3 Definition and Types of Biotic parameters ...
27
i. Herbivory ...
27
ii. Biofilm ...
27
iii. Epiphytism ...
28
1.4.1 Introduction ...
28
1.4.2 Chemical Defence ...
29
i. Oxidative bursts ...
30
ii. Halogenation of volatile and non-volatile compounds ...
30
iii. Distance signaling...
30
1.4.3 Microbial Defence...
31
1.5 Part – V – Consequence of Holobiont break-up - Macroalgal Disease ...
32
1.5.1 Introduction ...
32
1.5.2 Definition ...
33
1.5.3 Algal diseases ...
33
1.5.4 Nature of pathogens ...
34
1.5.5 Mechanisms of action of pathogen ...
35
1.5.6 Impact of disease on the aquaculture sector ...
36
1.6 Context and Aims of the Thesis ...
38
2. Chapter I - Defence on Surface: Macroalgae and their associated-microbiome ...
42
2.1 Abstract ...
44
2.2 Introduction ...
45
2.3 Seaweed surface-microbe interactions – biofilm development...
46
2.3.1 Seaweed Surface: as a substratum...
46
i. Cell wall structure ...
47
ii. Surface topographical features...
47
2.4 Macroalgae defence ...
47
2.4.1 Removal of surface layers ...
47
2.4.2 Production of Reactive Oxygen Species ...
48
2.4.3 Antimicrobial compounds ...
48
2.5 Microbiome on the surface ...
50
2.5.1 Bacteria ...
50
2.5.2 Fungi ...
57
2.5.3 Microalgae ...
60
2.5.4 Virus ...
61
2.6 Role of Microbiome: Microbial Interactions and their Hosts Response ...
62
2.6.1 Quorum Sensing ...
63
2.6.2 Quorum Quenching ...
65
2.6.4 Negative Effects: Pathogenism ...
68
i. Spatial effects ...
69
ii. Temporal Effects ...
70
2.7 Future Perspectives ...
70
2.8 References ...
72
3. Chapter II - Screening of surface-associated bacteria from the Mexican red alga Halymenia floresii for Quorum Sensing activity ...
74
3.1 Abstract ...
78
3.2 Introduction ...
80
3.3 Materials and methods ...
81
3.3.1 Algal material ...
81
3.3.2 Isolation of surface-associated bacterial strains ...
82
3.3.3 Characterization of selected bacterial strains ...
82
i. Morphological and biochemical characterization ...
82
ii. Molecular characterization ...
82
iii. Phylogenetic analyses ...
83
3.4 Screening of the selected bacterial for QS signals - Reporter assay ...
83
3.4.1 Reporter strains ...
83
3.4.2 Cross-feeding assay...
83
3.4.3 Bioluminescent assay ...
84
3.5 Statistical analysis ...
84
3.6 Results ...
84
3.6.1 Isolation and identification of surface-associated bacteria ...
85
i. Morphological and biochemical characterization ...
85
ii. Molecular characterization ...
88
a. Alphaproteobacteria ...
88
b. Gammaproteobacteria ...
88
c. Bacteroidetes ...
88
d. Firmicutes ...
89
3.6.2 Detection and screening of QS signal production ...
90
3.7 Discussion ...
96
3.7.1 Isolation and identification of selected surface-associated bacteria of H. floresii ...
96
3.7.2 Detection and screening of QS signals...
98
i. Cross-feeding assay...
98
3.8 Conclusions ...
100
3.9 Future perspectives ...
100
3.10 Acknowledgements ...
101
3.11 References ...
101
4. Chapter III - Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta) ...
103
4.1 Abstract ...
107
4.2 Introduction ...
109
4.3 Materials & Methods ...
111
4.3.1 Algal material ...
111
4.3.2 Optimization of Selective Extraction of surface-associated metabolites ...
111
i. Surface-associated metabolites (DIP) extraction – Determination of Immersion solvent and Immersion period ...
111
ii. Whole-Cell Metabolites (WCM) extraction ...
112
4.3.3 Quorum Quenching activity by Bioluminescent reporter assay...
113
i. Bacterial strains – Isolation from the surface of H. floresii ...
113
ii. Identification – Taxonomic affiliation of the surface-associated bacteria ...
113
iii. Bioluminescence – Reporter Assay ...
113
a. Preparation of supernatants ...
113
b. Reporter strain ...
114
c. Bioluminescent assay ...
114
4.3.4 Untargeted Metabolomic profiling - Identification of the compounds by LC-MS ...
114
i. LC-MS conditions ...
114
ii. Pre-processing and processing of data: XCMS Online and the Madison Metabolite Consortium Database ...
115
4.4 Statistical Analysis ...
115
4.5 Results ...
115
4.5.1 Algal material - Culture of H. floresii ...
115
4.5.2. Optimization of Selective Extraction of surface-associated metabolites ...
116
i. DIP Extraction...
116
ii. WCM Extraction ...
119
4.5.3. Quorum Quenching activity of DIP extracts by Bioluminescence – Reporter Assay ...
119
i. Identification – Taxonomic affiliation of bacterial strains ...
119
ii. Bioluminescence – Reporter Assay ...
120
4.6 Discussion ...
124
4.6.1 Selective extraction of surface-associated metabolites ...
124
4.6.2 Influence of DIP n-hexane extract on bacterial communication ...
126
4.6.3 Untargeted Metabolomic Profiling ...
127
4.7 Acknowledgements ...
129
4.8 References ...
129
5. Chapter IV - Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method ...
132
5.1 Materials and Methods ...
134
5.1.1 Algal material ...
134
5.1.2 Bacterial isolates ...
134
5.1.3 Tip bleaching assay with single epibacterial strains ...
134
5.1.4 Extraction of HomoSerine Lactones (HSLs) from the ‘significant pathogen’ ...
135
5.1.5 Chromatographic conditions – LC-MS QTOF analysis ...
136
5.2 Results ...
137
5.3.1 Tip bleaching assay with single epibacterial strains ...
137
8.3.2 Identification of homoserine lactones (HSLs) from the ‘significant pathogen’ ...
140
5.3 Discussion ...
143
5.5 Conclusion and perspectives ...
145
6. Chapter V - Screening Halymenia floresii secondary metabolites which underly its surface defence mechanisms ...
147
6.1 Abstract ...
153
6.2 Introduction ...
155
6.3 Materials and Methods ...
157
6.3.1 Algal material and extractions ...
157
6.3.2 LC-MS conditions and data pre-processing ...
158
6.3.3 LC-MS data processing – Madison Metabolomics Consortium Database...
159
6.3.4 Data post-processing - SMILES ... 160
6.3.5 Data Validation & Interpretation - Statistical Analysis ... 161
6.4 Results ...
163
6.4.1 LC-MS analyses ...
163
6.4.2 Identifying the H. floresii secondary metbolites ...
163
6.4.3 H. floresii secondary metabolites ...
172
6.4.4 Hydrophobicity of the secondary metabolites ...
176
6.6 Conclusion ...
183
6.7 Acknowledgements ...
184
6.8 References ...
184
7. General Discussion ...
186
7.1 Origin of the Thesis ...
187
7.2 Holobiont: Halymenia floresii and the associated bacterial community ...
187
7.3 The partner: associated bacteria ...
188
7.3.1 Characterisation of bacterial community ...
188
7.3.2 Influence of the epibacterial community...
190
7.4 The Host - H. floresii ...
191
7.5 Interactions among bacteria and the Host’s Interference ...
193
7.5.1 Analysis of positive relation– symbiotism ...
194
7.5.2 Putative opportunistic pathogenicity ...
194
7.6 Aquaculture/Disease/Global Change ...
195
7.7 Future perspectives ...
196
7.8 Future possible experimentations ...
200
8. Experimentation Setup ...
203
8.1 Media composition ...
203
8.2 Chemicals ...
203
8.3 Antibiotics ...
204
8.4 DNA Extraction Kits ...
204
8.5 PCR Amplification – 16S ...
205
List of communications
Oral International
A Abdul Malik S., Gamboa A., Bedoux G., Garcia Maldonado J. Q., Bourgougnon N., & Robledo D. Composition, symbiosis and pathogenesis, an equilateral relationship of the surface-associated microbiome of the cultivated Mexican Rhodophyta, Halymenia floresii, 67th Annual Meeting of The British Phycological Society (07 January – 10 January, 2019) Scottish Association of Marine Sciences, Oban, Scotland.
A Abdul Malik S., Bedoux G., Robledo D., Garcia Maldonado J. Q., Freile-Pelegrín Y., & Bourgougnon N. Chemical defence by allelopathic active metabolites on the surface of cultivated Mexican Rhodophyta Halymenia floresii against biofouling, 23rd International Seaweed Symposium (28 April – 03 May, 2019),
International Seaweed Association, Jeju, Korea.
Oral National
A Abdul Malik S., Caamal-Fuentes E., Ávila-Velázquez V., Bedoux G., Bourgougnon N., Robledo D. Involvement of allelopathic active metabolites on the surface of the cultivated Halymenia floresii in the biofouling phenomenon, Société Phycologique de France, Paris.
List of Figures (outside the chapters)
Fig. 1 Worldwide macroalgal production (%) under aquaculture per species and countries-year 2014
(Source: FAO, 2016) (Buschmann et al. 2017)... 5
Fig. 2 Coastline of the Yucatan Peninsula including the two outstanding marine ecosystems, Gulf of Mexico and Caribbean Sea ... 7
Fig. 3 Spatial distribution and Genus richness of Rhodophyta clade worldwide as depicted in (Keith et al. 2014). ... 9
Fig. 4 Schematic cell wall of red algae with characteristic polysaccharides (Stiger-Pouvreau et al. 2016). ... 11
Fig. 5 Types of carrageenans (Jiao et al. 2011)... 13
Fig. 6 Floridean Starch ... 14
Fig. 7 Floridoside ... 15 Fig. 8 Isofloridoside ... 15 Fig. 9 Digeneaside ... 15 Fig. 10 R-Phycoerythrin ... 17 Fig. 11 R-Phycocyanin ... 17 Fig. 12 Allophycocyanin ... 17
Fig. 13 Halymenia floresii, (Clemente & Rubio) C.A. Agardh (Rhodophyta, Halymeniales), from the Yucatan peninsula, Mexico ... 19
Fig. 14 Observation of the surface of H. floresii under different microscopes; (a, b - cross and longitudinal section view of H. floresii under ‘Phase Contrast’ microscope; c, d - the surface of H. floresii under ‘Epifluorescence microscope’ (40x and 400x); e, f – the surface of H. floresii under ‘Scanning Electron Microscope’ (10 µm and 1 µm) ... 21
Fig. 15 Main abiotic and biotic factors influencing the ecology and physiology of macroalgae (Lalegerie et al. 2020) ... 24
Fig. 16 Defence mechanism of marine macroalgae against epibionts (da Gama et al. 2014) ... 29
Fig. 17 Macroalgal diseases in different algal species. a – rot in Pyropia (Kim et al. 2014); b – Red-rot in Pythium porphyrae mycelium (Kim et al. 2014); c – bleached D. pulchra (Zozaya-Valdés et al. 2016); d – bleached K. alvarezii green (Arasamuthu and Patterson Edward 2018); e – bleached coralline algae (downloaded from http://coralreefdiagnostics.com/); f – bleaching in H. floresii ... 34
Fig.18 Isolation of the epibacterial strains by swabbing, streaking, and sub-culturing ... 76
Fig. 19 Identification of the isolates by biochemical and molecular characterization ... 76
Fig. 20 Reporter assays to screen the isolates for QS activity ... 77
Fig. 21 Extraction of surface-associated metabolites by the method of DIP ... 105
Fig. 22 Evaluation of Quorum Quenching activity by bioluminescent assay using E. coli pSB406 ... 105
Fig. 23 Identification of the surface-metabolites by untargeted metabolomic approach ... 106
Fig. 25 Pre-processing of the raw data (.mzXML) in XCMS Online platform ... 150 Fig. 26 Processing of the data in Madison Metabolomics Consortium Database (MMCD) ... 151 Fig. 27 Metabolite organization to yield the corresponding structural and functional descriptors of the predicted metabolites ... 151 Fig. 28 Statistical analysis to validate the data performed in ‘R Studio’ ... 152 Fig. 29 Different classes of bacteria (%) isolated from the surface of H. floresii ... 189 Fig. 30 Scanning electron micrographs of the surface of H. floresii; a – apical fronds; b – mid-thallus; c – stalk. ... 190 Fig. 31 Count of Metabolites (‘41’) grouped based on their Activity/Biochemical classes (ATB –
Antibiotics; ATC - Anti-cancerous; ATF – Antifungal; ATI - Anti-inflammatory; ATO - Anti-oxidant; ATP – Antiproliferative; ATV – Antiviral; INH – Inhibitors; DIV – Various; FUR – Furans; HAL – Halogenated; HOR – Hormones; IME - Intermediary Metabolites; KET – Ketones; LIP – Lipids; NUC – Nucleotides; PIG – Pigments; SAC - (Poly)-Saccharides; VIT - Vitamins) ... 191 Fig. 32 Bioactive potential of H. floresii identified by untargeted metabolomics study ... 198
List of Figures (in the chapters)
Chapter IFig. 1 Bioactive compounds produced by the red, green and brown macroalgae ... 49 Fig. 2 Bioactive metabolites from the macroalgal associated bacteria ... 56 Fig. 3 Bioactive metabolites from the macroalgal associated fungi ... 59 Fig. 4 Diatoms embedded on the extracellular matrix of H. floresii (left) and associated with the
macroalgal surface bacteria (right) ... 61 Fig. 5 Different homoserine lactones with differing chain length produced by the bacteria ... 64
Chapter II
Fig. 1 Induction of bioluminescence in the reporter E.coli pSB406 by Vibrio owensii (B3IM); Maribacter sp., (B9IM); Pseudoalteromonas arabiensis (B4BC); P. tetraodonis (B6BC) and V. owensii (B7BC) No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm ... 92 Fig. 2 Induction of bioluminescence in the reporter E.coli pSB406 by Tenacibaculum sp., (B9BC);
Spongiimicrobium sails (B9CC); Aquimarina sp., (B9.1CC); Alteromonas sp., (B12CC) and
Pseudomonas aeruginosa PA01 (positive control). No. of cycles (30 minutes per cycle) plotted against
relative light units/optical density at 600 nm ... 93 Fig. 3 Induction of bioluminescence in the reporter E.coli pSB406 by Pseudoalteromonas sp., (B5BC); North sea bacterium (B6.1BC) and Alteromonas marina (B16CC). No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm. Culture period, in hours, of the isolates were denoted as ‘h’. ... 94
Fig. 4 Induction of bioluminescence in the reporter E.coli pSB406 by Ruegeria lacuscaerulensis (B9CC); Uncultured bacterium clone (B9.2CC) and Erythrobacter sp., (B14CC). No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm. Culture period, in hours, of the
isolates were denoted as ‘h’. ... 95 Fig. 5 Induction of bioluminescence in the reporter E.coli pSB406 by Roseobacter sp., (B13CC) and
Thalassococcus sp., (B20CC). No. of cycles (30 minutes per cycle) plotted against relative light
units/optical density at 600 nm. Culture period, in hours, of the isolates were denoted as ‘h’. ... 96
Chapter III
Fig. 1 Selective extraction of surface-associated and whole-cell metabolites. Diagram of extracts analyses and data processing of cultivated H. floresii (CC). ... 112 Fig. 2 Epifluorescence micrographs showing the cortical cells of H. floresii after being dipped in n-hexane for 10s; 20s; 30s; 40s; 50s and 60s and positive control treated in exactly the same way with sterile seawater for 30s. ... 117 Fig. 3 Number of intact and lysed cells vs. immersion period (in seconds) after dipping in n-hexane counted using ImageJ software. The y-axis represents the Means ± SE of epifluorescence micrographs of H. floresii at ‘5’ different angles (n=3). ... 118 Fig. 4 (a-d) Quorum quenching activity of the DIP extract on the chosen phylotypes. a –Vibrio owensii; b – Tenacibaculum sp.; c – Maribacter sp. and d – Aquimarina sp. No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm. ... 121 Fig. 5 TIC of DIP_n-hexane (red) and WCM_n-hexane (blue) crude extract after retention time correction by XCMS Online platform. ... 122 Fig. 6 LC-MS spectrum of surface extract showing bromine isotopic pattern (red arrow marks) a
characteristic of halogenated metabolite. ... 128
Chapter IV
Fig. 1 Schematic illustration of HSLs extraction process………..136 Fig. 2 Risk of thallus tip bleaching in Halymenia floresii after inoculation of ‘25’ bacterial strains relative to control thalli without such inoculation. No. of replicates (n=6) were assigned for each isolate. Error bars ± in % ... 138 Fig. 3 Chromatogram of V. owensii HSL extract pre-processed in XCMS Online platform ... 141 Fig. 4 Chromatogram showing the presences of C4 HSL in the standard and V. owensii extract at a
retention time of 6.2 to 7.0 minutes ... 141 Fig, 5 Mass Spectrum showing the fragments of C4 HSL in the standard and V. owensii extract as 102.05 (lactone ring); 172.09 (M+H)+ and 194.08 (M+Na)+. ... 142
Fig. 6 Chemical structures corresponding to the mass spectrum at 102.05 (lactone ring); 172.09 (M+H)+
Chapter V
Scheme I Schematic workflow of untargeted metabolomic approach on Halymenia floresii ... 162 Fig. 1 Metabolite counts of the Whole-cell metabolites (WCM); surface-associated metabolites (DIP) and common metabolites (CMN) plotted against Activity/Biochemical classes (symbols are from Table 1).164 Fig. 2 Left: Relative class occurrence (%) of the Whole-cell metabolites (WCM); surface metabolites (DIP) and common metabolites (CMN) plotted against Activity/Biochemical classes (symbols as in Fig. 1). Right: Metabolic count (A) and Relative Class occurrence (B) in % of the Whole-cell metabolites (WCM=416) and common metabolites (CMN=41) plotted against Activity/Biochemical classes ... 165 Fig. 3 Dendrogram resulting from the hierarchical cluster analysis (HCA) of ’41’ metabolites ordered into ‘8’ clusters (I -VIII) at a Ward distance of 1.0. The ‘26’ compounds for which a bioactivity has been reported are green labelled ... 172 Fig. 4 Clustering of the H. floresii ‘41’ common metabolites (CMN) according to chemical structures (retrieved from Data Warrior) calculated from their SMILES strings. Numbers correspond to Table 2 ordering. ... 174 Fig. 5 Entanglement of chemical (left) and lipophilicity (right) dendrograms. The level of entanglement is 0.535 (intermediate between 0 and 1) ... 176 Fig. 6 Lipophilicity of the H. floresii secondary metabolites as a function of the compound's bioactivity. Octanol-water partition coefficients are calculated from the XLOGP3 method ... 177
List of Tables
Chapter ITable 1 List of bacteria isolated from the ‘3’major phyla of macroalgae ... 51 Table 2 List of fungi isolated from the ‘3’major phyla of macroalgae ... 57 Table 3 List of microalgae isolated from the macroalgae ... 60 Table 4 List of viruses isolated from the macroalgae ... 62
Chapter II
Table 1 Motility, Gram stain and Biochemical characteristics of the isolates ... 86 Table 2 Halymenia floresii surface-associated bacteria; * - Strain with <97% similarity. ... 89 Table 3 Screening of the isolates based on QS signals production; * - Strain with <97% similarity. ... 91
Chapter III
Table 1. Concentration of the H. floresii, DIP extracts with different solvents and WCM extracts with n-hexane ... 118 Table 2. Taxonomic affiliation of the isolated phylotypes. ... 119 Table 3. List of common metabolites in DIP and WCM n-hexane extracts proposed by the MMCD mass bank ... 123
Chapter IV
Table I Epibacterial strains isolated from Halymenia floresii tested for their pathogenicity and/or non-pathogenicity in tip bleaching assay ... 139
Chapter V
Table 1 Codes assigned, based on the bioactivity (functional descriptors) and/or chemical class (chemical descriptors), to the metabolites (DIP and WCM) proposed by the massbank ... 159 Table 2 Codes assigned to the ‘41’ CMN metabolites, names of the putatives, raw/chemical formula, confidence level (∆), and actual mass as proposed by the database MMCD, experimental m/z features (Exp m/z), retention time (RT in minutes) and maximum intensity resulted from the XCMS Online platform, the corresponding Bioactivity /Biochemical classes of putatives (BIO) and their LOGP3 octanol water partition coefficients (XLogP3-AA). ND – Not Detected. ... 166
List of Abbreviations
AHLs – Acylated Homoserine Lactones
AHPND – Acute Hepato Pancreatic Necrosis Disease AIs - AutoInducers
AIPs – Auto Inducing Peptides ANOVA – Analysis of Variance BC – Beach-cast
BHL - N-Butryl-DL-Homoserine Lactone BLAST – Basic Local Alignment Search Tool CC – Cultivar Chamber
CFB – Cytophaga-Flavobacterium-Bacteroides CMN - Common
DCM - Dichloromethane
DIN – Dissolved Inorganic Nitrogen EDTA – Ethylene Diamine Tetraacetic acid HHL - N-Hexanoyl-DL-Homoserine Lactone IMTA – Integrated Multi Trophic Aquaculture LB – Luria Broth
MMCD – Madison Metabolomic Consortium Database MA – Marine Agar
MB – Marine Broth MeOH – Methanol
NCBI – National Center for Biotechnology Information OOHL - N-(3-Oxooctanoyl)-L-homoserine lactone PCR – Polymerase Chain Reaction
rRNA – ribosomal Ribonucleic acid RLU – Relative Light Unit
SMILES – Simplified .sdf - Structure-Data File
TAE – Tris base, Acetic acid, EDTA QS – Quorum Sensing
QSI – Quorum Sensing Interference QQ – Quorum Quenching
UV – Ultra-Violet
WCM – Whole-Cell Metabolites
Genesis of the project
In macroalgal cultivation, epiphytism and fouling are the major problems, severely reducing the productivity and cost efficiency of a diverse range of open-water and on-shore farms. Antifouling practices predominantly include the use of copper-based antifoulant coating, in combination with practical fish husbandry and site management practices. With rapid expansion of the aquaculture industry, coupled with the tightening of legislation on the use of antifouling biocides, the problems of aquaculture biofouling are increasing. The development of environmentally safe antifouling substances is urgently needed. In such a circumstance, during the experimental cycle of culture of Mexican Rhodophyta, Halymenia floresii (Clemente) C. Agardh cultivated in experimental Integrated Multi-trophic Aquaculture systems (IMTA) at onshore tanks, it was observed that, contrary to the tank cultures with Eucheuma, the walls and the propellers remained clean of any exogenic algae colonisation. The presence of H. floresii appeared to limit the establishment of opportunist green algae (Cladophora, Chaetomorpha or Ulva) and the colonisation of barnacles usually disturbing the cultures. Furthermore, the surface of the alga was remarkably free from settlement by fouling organisms. This ecological phenomenon could reveal the release of allelopathically active compounds interfering with the settlement and growth of competitors.
The ECOS Nord project is an international scientific cooperation project between FRANCE and MEXICO with the aim of upgrading the macroalgae by combining innovative biotechnologies and process for human health. This project brings together the Laboratory of Marine Biotechnology and Biochemistry
(LBCM), University of South Brittany (UBS), FRANCE and the research team of the Department of
Marine Resources integrated into The Center for Research and Advanced Studies of the National
Polytechnic Institute (CINVESTAV), MEXICO. With this perspective of ECOS Nord, the objective of
this project was proposed to better understand the ecological phenomenon behind the exceptional observation in onshore tanks of H. floresii culture.
1. General Introduction
1.1 Part I – Introduction to Macroalgae
1.1.1 Macroalgae
Approximately 70% of Earth’s surface is occupied by a continuous stretch of seawater with an average depth of 5 km (Baweja et al. 2016) with a rich source of both biological and chemical diversity. Oceans support floating forests with variable sea plants and animals, where marine vegetation is considered to be more primitive and richer than land-based plants. Among them, macroalgae (seaweeds) are important marine resources playing a major role in supporting the rich biodiversity of the sea. Macroalgae constitute a diverse polyphyletic group of predominantly marine, multicellular, photosynthetic, chlorophyll “a” containing, eukaryotic organisms with simple reproductive structures and found from the intertidal zone to 300 m deep (Levine 2016).
1.1.2 History and Classification
Most algae are photosynthetic organisms that acquired the trait some 1.8 billion years ago after an unknown non-photosynthetic unicellular eukaryote engulfed or was invaded by a photosynthetic cyanobacterium similar to Gloeomargarita. This trait ultimately resulted in a cell containing a photosynthetic plastid surrounded by two membranes with highly reduced cyanobacteria derived genome. This event, known as the primary endosymbiosis, gave rise to the extant Archaeplastida, which includes the Glaucophyta, Rhodophyta (red algae), and Chloroplastida (green algae and land plants) (Tirichine and Bowler 2011; Leliaert et al. 2012; Adl et al. 2019). Brown algae have a very different evolutionary history, as they have acquired the ability to achieve photosynthesis through a secondary endosymbiosis event. The other lines such as Cryptophytes, Haptophytes, Golden brown algae and Dinophytes, are therefore eukaryotes with no direct relationship with terrestrial plants. The lines resulting from secondary or even tertiary endosymbioses between a primitive non-photosynthetic eukaryote and a unicellular red alga include the Ochrophytes within Stramenopiles, Cryptophytes and Haptophytes lines. Derived from endosymbiosis with a green unicellular alga, the lines are grouped within Euglenophyceae and Rhizaria (Niklas and Kutschera 2010). Such events involved a second heterotrophic eukaryote that engulfed not a cyanobacterium but a green or red photosynthetic eukaryote and resulted in plastids with three or four membranes around them.
The customary notion of macroalgae actually encompasses a polyphyletic complex within eukaryotes (Boudouresque 2015). The evolutionarily diverse macroalgae are found in two kingdoms,
Plantae and Chromista, and four phyla, Charophyta (Chara), Chlorophyta (green), Rhodophyta, and Ochrophyta (brown). Most marine macroalgae belong to phylum Ochrophyta, class Phaeophyceae, and to phylum Rhodophyta. A few marine macroalgae occur in the phylum Charophyta and about 600 large green algal taxa occur in the phylum Chlorophyta. Macroalgae in these four clades have more fundamental (e.g. cytological, chemical, life histories) differences between one another than with the vascular plants (Dawes 1998). Dring (1982) listed 900 species of green algae, 997 brown algae, and 2540 red algae worldwide. These advancements in phycology have resulted in major changes in the classification of algae (Schneider and Wynne 2007, 2013; Wynne and Schneider 2010). Thus, the number of macroalgal species has increased. Guiry and Guiry (2020) listed 4885 species in the Charophyta and 6759 in the Chlorophyta, 2057 species in the class Phaeophyceae, and 7303 species in the Rhodophyta. In W.R. Taylor’s 1957 flora
Seaweeds of the Northeastern Coast of North America, he listed 170 genera (37 green, 57 brown, 76 red
algae) and 401 species (95 green, 142 brown, and 164 red algae), while Mathieson and Dawes (2016) have described 248 genera (57 green, 89 brown, 102 red) and 535 species (146 green, 180 brown, and 209 red taxa) for the same geography (Dawes 2016). As multicellular eukaryotes, the classification of marine macroalgae are made according to their photosynthetic pigments like green (chlorophyll α and β), red (phycoerythrin) and brown (xanthophyll/fucoxanthin) (Sudhakar et al. 2018).
1.1.3 Ecology
Macroalgae are ecologically important primary producers, competitors, and ecosystem engineers. They play a vital role in ecosystems as foundation species by providing habitat for higher trophic levels (Fulton et al. 2019). They constitute one of the important biotic components and play a central role in the different ecosystem in coastal habitats ranging from kelp forests to coral reefs (Gattuso et al. 2006; Gachon
et al. 2010; Keith et al. 2014; Singh and Reddy 2016; Krause-Jensen et al. 2018). Seaweeds serve as the
base of the marine food webs and are a direct food source for sea urchins and fish. In addition, they provide shelter and reproductive grounds for fish, invertebrates, birds, and mammals. Seaweeds along with coral animals are the dominant benthic organisms whose relative abundance is often used as an indicator of ecosystem health (Baweja et al., 2016).
The distribution of seaweeds depends upon many factors such as physical (substrate, temperature, light quality and quantity, dynamic tidal activity, winds, and storms), chemical (salinity, pH, nutrients, gases, and pollution level), and biological (herbivores, microbes, epiphytes, endophytes, symbionts, parasites, and diseases). Indeed, as sessile organisms, macroalgae have developed effective mechanisms to survive many biotic and live in a variable, extreme, and hostile abiotic environmental and stress conditions. As a result, these beings are able to produce a wide range of compounds called "secondary metabolites", like pigments, vitamins, phenolic compounds, sterols, other bioactive agents and some extremely relevant
biochemical mechanisms with biomedical and agricultural applications (Ioannou and Roussis 2009; Stengel
et al. 2011; Lalegerie et al. 2020).
1.1.4 Valorization of Macroalgae
Marine algae contain significant quantities of vitamins, minerals, dietary fibers, proteins, polysaccharides, and various functional polyphenols while nutrient contents can vary with species and, geographical location, season, and temperature. With such potential, macroalgae are subjected to numerous valorizations, particularly in the fields of human and animal nutrition. As human food, the valorization is direct. More than 600 species of seaweeds are edibles and used as vegetables in the world. Their nutritive value may vary depending on the geographic location, the season of the year, the growth stage, part of the seaweed harvested, etc. (Leandro et al., 2019). Thus, to assure the nutritional value of seaweeds, they need to be evaluated before being used as supplements. Seaweeds are industrially processed to extract thickening agents such as alginate, agar, and carrageenan cell wall polysaccharides or used, generally in dried powder form, as an animal-feed additive (Delaney et al. 2016). According to FAO (2018), some 221 species of seaweed are of commercial value and about ten species, such as brown seaweed (Saccharina japonica,
Undaria pinnatifida, and Sargassum fusiforme); red seaweed (Porphyra spp. Eucheuma spp. Kappaphycus alvarezii and Gracilaria spp.); and green seaweed (Enteromorpha clathrata, Monostroma nitidum and Caulerpa spp.). Red macroalgae, such as Gelidium, Gracilaria and Pterocladia, are important for human
consumption and other uses, mainly as a binder in food products as well as the bacterial substrate in laboratories. Eucheuma and Kappaphycus are essential for the manufacture of carrageenan, used in cosmetics, food processing and industrial usage (FAO, 2018).
Macroalgae and their uses have a long history. Human and macroalgal interactions seem to date back to the Neolithic period and over the years the industrial use of macroalgae has shifted as the potential of the future industry has been viewed as being larger than its actual scale. From exploiting beach-cast macroalgae as fertilizer to their higher value as a source of polysaccharides and their technical applications in agronomics, cosmeceuticals, nutraceuticals and pharmaceuticals (Galland-Irmouli et al. 1999; Smit 2004; Fleurence et al. 2012; Buschmann et al. 2017), the potential of macroalgae has raised the need of their extensive cultivation. By far the largest producers are China and Indonesia followed by the Philippines, Korea, Japan, Malaysia, and Zanzibar in Asia whereas in the Americas only Chile has appeared in the farming statistics tables (Fig. 1). In contrast, Europe, Canada, and Latin America, the macroalgal industries stilly rely on natural harvest (Rebours et al. 2014).
Fig. 1 Worldwide macroalgal production (%) under aquaculture per species and countries-year 2014 (Source: FAO, 2016) (Buschmann et al. 2017)
According to FAO statistics (FAO, 2014, 2016), of the top seven most cultivated seaweed taxa, three were used mainly for hydrocolloid extraction: Eucheuma spp. and Kappaphycus alvarezii for carrageenans, and Gracilaria spp. for agar; Saccharina japonica (formerly Laminaria japonica), Undaria
pinnatifida, Porphyra/Pyropia spp. and Sargassum fusiforme were most important in human food usage
(Fig. 1). The main producing countries were China, Indonesia, and the Philippines, which were also those that cultivated the greatest diversity of seaweed species. While seaweed consumption in South-east Asia has been common and traditional and has depended on taste and price, seaweed use as food in non-Asian European and USA markets has considered additional parameters such as nutritional value and ‘food for health’, with a strong consumer preference towards organic, sustainable and fair trade products (representing low impacts both on the environment and biodiversity) (Chapman et al. 2015; Gomez Pinchetti and Quintana 2016). However, the global market share of seaweed farming production used for food and ‘other uses’, i.e. other than for hydrocolloids, is still below 1% of the total biomass production (Buschmann et al. 2017). In 2014, about 28.5 million tonnes of seaweeds and other algae were harvested for direct consumption or further processing for food (traditionally in Japan, the Republic of Korea, and China) or for use as fertilizer and in pharmaceuticals, cosmetics and other purposes (FAO, 2016). In 2015 the total production of seaweed was 30.4 million tonnes, with the culture and capture sectors responsible for 29.4 million tonnes and 1.1 million tonnes, respectively (FAO, 2018).
The global seaweed industry is worth more than USD 6 billion per annum (approximately 12 million tonnes per annum in volume) of which 85% comprises food products for human consumption. The seaweed-derived extracts make up almost 40% of the world’s hydrocolloid market in terms of foods (FAO, 2018). The increasing worldwide demand for raw materials raises the issues of sustainability, access, or availability. Aquaculture presents an alternative to ensure the availability of this resource and improve its homogeneous quality. In addition, advances in the development of seaweed cultivation can compensate the wild production and collection, and thus improving productivity and quality of seaweed biomass. Fulfilling aquaculture's growth potential requires responsible technologies and practices. Sustainable aquaculture should be ecologically efficient, environmentally benign, product-diversified, profitable, and societally beneficial. This ultimately paved a way for the emergence of innovative aquaculture practices and one such method is Integrated Multitrophic Aquaculture (IMTA), which can help address many of the environmental impacts of aquaculture. It also aims at a balanced ecosystem-based management approach to aquaculture (Chopin 2013, 2017).
IMTA aims to be an ecologically balanced aquaculture practice that co-cultures species from multiple trophic levels to optimize the recycling of farm waste as a food resource (Troell et al. 2009). By integrating fed aquaculture (finfish or shrimp) with inorganic and organic extractive aquaculture (macroalgae and invertebrates), IMTA can significantly increase the sustainability of aquaculture. The IMTA system is driven only by sunlight, natural inorganic nutrients, as well as carbon dioxide and provides efficient food provisions, nutrient extraction, and climate regulating services to the marine ecosystem (Cottier-Cook et al. 2016). The land-based IMTA operations have also been advocated as a way of controlling diseases and their transmission, though some concerns have been expressed that multiple species on the site might increase the risk of disease transmission (Chopin 2013). Still, this raises a question on density rather than the diversity of the species.
1.1.5 Macroalgae in Mexico
The coastline of Mexico extends for 11 769 km of which 72% is located at the Pacific Coast and 28% at the Gulf of Mexico and the Caribbean coast (Robledo 2006). They include five geographic regions in temperate to tropical latitudes, Baja California, Gulf of California, Tropical Pacific, Gulf of Mexico, and the Mexican Caribbean with distinctive physiographic, geological, and climatic conditions make possible the existence of a very diverse algal flora. The Gulf of Mexico and the Caribbean Sea (Fig. 2) are two outstanding marine ecosystems joining in the Yucatan channel. Even though the Yucatan Peninsula (known as a biotic province) occupies 17.4% of the total of the Mexican coast, the investigation of marine resources has not been intensively investigated (Pech-Puch et al. 2020). This particular geographical situation promotes the existence of high diversity and abundance of different marine algal species, which represent
a potential source of bioactive compounds and food (Morales et al. 2006; Vázquez-Delfín et al. 2019; Alemañ et al. 2019).
Fig. 2 Coastline of the Yucatan Peninsula including the two outstanding marine ecosystems, Gulf of Mexico and the Caribbean Sea
Macroalgae of Mexico are very diverse. All three major types, green, brown, and red macroalgae show a greater species richness. In Mexico, 2,702 macroalgal species were described including freshwater (1,102 species) and marine (1,600 species) and nearly 800 species were reported in the Yucatan Peninsula. The diversity of habitats in the Yucatan Peninsula favours the most diverse red algae group of the region, with approximately 350 recorded species, distributed in about 15 taxonomic families (Dreckmann 1998; Ortega et al. 2001; Mendoza-González et al. 2007, 2016; Wynne 2017). In spite of their high diversity algal drifts, however, in this region of Mexico pose a challenge rather than their economic importance. In Yucatan, the most abundant families that drift for both the number of species and the amount of biomass were Gracilariaceae (10 species), Rhodomelaceae (6 species) and Solieriaceae (3 species) (Nuñez Resendiz
et al. 2019).
In Mexico, for about 50 years, the commercial exploitation of phycocolloid-producing algae has occurred continuously and they exported 439 tonnes of macroalgae in 2009 (Robledo et al. 2013). The commercial harvesting of natural populations of macroalgae includes three species of Rhodophyta,
Gelidium robustum, Chondracanthus canaliculatus, and Gracilariopsis lemaneiformis (Robledo 2006) and
one species of Phaeophyta, Macrocystis pyrifera (Robledo et al. 2013).
The Peninsula of Yucatan has 1,940 km of coastline, reportedly with the rich diversity of macroalgae. Rhodophyta has the greatest diversity on the coasts of Yucatan. A large variation in the chemical composition of the macroalgae from Yucatan was observed, particularly in soluble carbohydrates, ash, and protein (Robledo and Freile-Pelegrín 1997) which encouraged the experimental cultivation of native and exotic species during the years of 2000-2010. The lower seawater temperature may explain the abundance and diversity of macroalgal species as well the cold and warm water currents influence the distribution patterns of marine macroalgae (Robledo 2006). The presence of structurally diverse secondary metabolites is invaluable due to its potential application in drug discovery (Stengel et al. 2011; Goulitquer
et al. 2012; Altmann 2017; Wells et al. 2017; Hernández-Bolio et al. 2019; Pech-Puch et al. 2020). Being
a most conspicuous component on the Yucatan coasts, red algal species are highly valued in the international market for their secondary metabolites (Nuñez Resendiz et al. 2019).
1.2 Part – II– Red Macroalgae 1.2.1 Introduction
Rhodophyta represents an ancient photosynthetic lineage of eukaryotes that arose through primary endosymbiosis, with a fossil record dating back over 1.2 billion years (Butterfield 2000). Presently the phylum contains about 7303 species (Guiry and Guiry 2020) of which 3% is freshwater taxa (Sheath and Cole 1984). Based on molecular and morphological studies, Saunders and Hommersand (2004) identified three subphyla in the Rhodophyta: Metarhodophytina, Rhodellophytina, and Eurhodophytina. With the subphylum Eurhodophytina containing macroalgae divided into two classes: Florideophyceae (6950 species) and Bangiophyceae (182 species). Florideophyceae regroups many orders: Ahnfeltiales (11 species), Ceramiales (2690 species), Corallinales (603 species), Gelidiales (235 species), Gigartinales (951 species), Gracilariales (238 species), Halymeniales (357 species), Nemaliales (281 species), Palmariales (45 species), and Rhodymeniales (410 species). Bangiophyceae is a taxonomic unit less diversified. Among the two orders constitutive of the class, the order Bangiales regroups 182 recognized species with specimens of the genera Bangia and Porphyra/Pyropia (Schneider and Wynne 2007; Verbruggen et al. 2010; Yoon et
al. 2016; Adl et al. 2019).
Many red algae are found in the intertidal and subtidal zones to depths of up to 40, or occasionally, 250 m, and of course, they have the ability to live at greater depths in the ocean than the members of other algal groups. They probably represent one of the oldest groups of eukaryotes and most diversified groups.
From a morphological point of view, the majority of Rhodophyta is filamentous (Antithamnion), pseudoparenchymatous (Palmaria), or parenchymatous (only in Bangiales, Porphyra/Pyropia). Ceramium and Polysiphonia present a vegetative and siphoned filamentous. Some species are calcified. Corallinales contain a form of calcite (calcium carbonate) and are important contributors to the formation of tropical reefs. Red macroalgae are found in tropical, temperate and arctic regions, but are generally the most abundant in temperate regions and in tropical regions on sandy or rocky substrates (Keith et al. 2014).The overall distribution of genus richness for Rhodophyta had a hotspot on the west coast of North America and also, they had the lowest genus richness in the tropics compared with higher altitudes (Fig. 3).
Red algae are considered as the most important source of many biologically active metabolites in comparison to the other algal class. The majority of macroalgal secondary metabolites has discovered in red macroalgae accounting for >1500 bioactives (Amsler et al. 2005; Maschek and Baker 2008; Wijesekara
et al. 2011; Stengel et al. 2011). Meanwhile, the red macroalgae are wild-harvested from 32 countries,
indicating their widespread popularity, with a total of 216 456 t harvested worldwide (FAO, 2014).
Fig. 3 Spatial distribution and Genus richness of Rhodophyta clade worldwide as depicted in (Keith et al. 2014).
1.2.2 Biology of Red Macroalgae
One of the oldest groups of eukaryotic algae, red algae are an ancient group of eukaryotic plants and the majority of them are filamentous (Polysiphonia), pseudoparenchymatous (Ceramium) or parenchymatous (Porphyra/Pyropia, Halymenia). Rhodophyceae vary considerably in their response to different environmental factors, having adapted to living in nearly freshwater to brackish and marine conditions. Members of red algae are found in the tropical, temperate, and arctic waters, but are most abundant in the temperate and tropical regions. The common features of red algae include eukaryotic cells, a complete lack of flagellar structure, food reserves of floridean starch, the presence of phycobiliproteins (details in “Pigments and Proteins”) chloroplasts without stacked thylakoids and no external endoplasmic reticulum (Baweja et al. 2016). The members of Rhodophyta can tolerate a wider range of light levels than any other groups of photosynthetic organisms and this is supported by the presence of additional accessory pigments together with chlorophyll a. These accessory pigments generally known as phycobiliproteins allow them to live in deep waters. The major product of photosynthesis in red algae is floridoside (O-α-d-galactopyranosyl-(1,2)-glycerol), isofloridoside digeneaside or floridean starch (details in ‘Starch’). Another very common feature of red algae is pit connections providing symplastic communication between cells (Lee 2008). Marine red algae contain significant quantities of vitamins, dietary fibers, proteins, polysaccharides, and various micro- and macro elements (Baweja et al. 2016).
i. Cell wall
The cell wall organization and composition of the Rhodophyta differ significantly from plants and other macroalgae. The cell wall is a rather well-ordered structure as observed by optical and electronic microscopy (Gordon-Mills et al. 1978; Craigie 1990). The cell wall is considered here as including polymeric materials originating from the metabolic activities of the algae. Most investigators recognize polysaccharides, proteoglycans, peptides, proteins, lipids, and associated inorganic constituents as components of the native cell wall of red algae. According to previous studies on biological function, the polysaccharides may be grouped with the more rigid structural (β-linked) glycans such as cellulose, mannans, and xylans, as well as with the more flexible and frequently sulfated glycans that comprise the matrix in which the skeletal fibers are embedded (Rees 1981; Baweja et al. 2016).
The wall of red algae is classically illustrated by two phases or matrices:
1) neutral lower crystalline form with cellulose that forms a weft-like mat (Dawes et al. 1961). Some xylan and mannan have been also characterized in the amorphous embedding matrix.
2) charged phase that contains sulphated galactan polymers, some of which are economically important phycocolloids (carrageenan or agar). The cell wall consists of rigid components such as microfibrils and a mucilaginous matrix. (Fig. 4) (Stiger-Pouvreau et al. 2016).
Fig. 4. The schematic cell wall of red algae with characteristic polysaccharides (Stiger-Pouvreau et al. 2016).
Under the optical microscope and in electron microscopic studies Gracilaria corticata reveals cells having thick cell walls that have microfibrils arranged in three distinct layers: (i) the inner-most electron-dense (glycoprotein domain), (ii) middle electron-translucent (amorphous matrix) and (iii) outermost electron-dense regions (fibrillar wall). In all three regions, microfibrils are arranged in parallel. The extracellular matrix consists of a cellulose microfibrillar network and an amorphous matrix of cellulose, sulphated galactans, and mucilage.
ii. Polysaccharides: Structural and Storage
a. Carrageenans
Red seaweeds contain large amounts of cell-wall polysaccharides, most of which are sulphated galactans. These galactans are generally built on repeated alternating l,3-linked α-galactopyranose and l,4-linked β-galactopyranose units and differ in the level and pattern of sulphation, in the substitution of
