Marine Algae Extracts: Processes, Products, and Applications

By Se-Kwon Kim

Designed as the primary reference for the biotechnological use of macroalgae, this comprehensive handbook covers the entire value chain from the cultivation of algal biomass to harvesting and processing it, to product extraction and formulation.

In addition to covering a wide range of product classes, from polysaccharides to terpenes and from enyzmes to biofuels, it systematically discusses current and future applications of algae-derived products in pharmacology, medicine, cosmetics, food and agriculture.

In doing so, it brings together the expertise of marine researchers, biotechnologists and process engineers for a one-stop resource on the biotechnology of marine macroalgae.

• Language: English
• Publisher: Wiley
• Release date: Feb 10, 2015
• ISBN: 9783527679591

Book preview

List of Contributors

Helena T. Abreu

ALGAplus– Produção e comercialização de algas e seus derivados, Lda., CIEMAR

Travessa Alexandre da Conceição

3830-196 Ílhavo

Portugal

Marzia Albenzio

University of Foggia

Department of the Sciences of Agriculture

Food and Environment (SAFE)

Via Napoli, 25

71122 Foggia

Italy

Sekar Ashokkumar

The University of Suwon

Department of Bioscience and Biotechnology

Hwaseong-si 45-743

Republic of Korea

Ashish Bhatnagar

Maharshi Dayanand Saraswati University

Algae Biofuel and Biomolecules Centre

Ajmer 305 009

India

Monica Bhatnagar

Maharshi Dayanand Saraswati University

Algae Biofuel and Biomolecules Centre

Ajmer 305 009

India

Dumitru Bulgariu

Alexandru Ioan Cuza University of Iasi

Department of Geology

Faculty of Geography and Geology

Iasi

Romania

and

Filial of Iasi

Collective of Geography

Romanian Academy

Iasi

Romania

Laura Bulgariu

Technical University Gheorghe Asachi of Iasi

Department of Environmental Engineering and Management

Faculty of Chemical Engineering and Environmental Protection

D. Mangeron, No. 73

700050 Iasi

Romania

Damien L. Callahan

Deakin University

Centre for Chemistry and Biotechnology

School of Life and Environmental Science

Burwood

Victoria 3125

Australia

and

Metabolomics Australia

The University of Melbourne

The School of Botany

Parkville

Victoria 3010

Australia

Mariangela Caroprese

University of Foggia

Department of the Sciences of Agriculture

Food and Environment (SAFE)

Via Napoli, 25

71122 Foggia

Italy

Luciana R. de Carvalho

Instituto de Botânica

Núcleo de Pesquisa em Ficologia

Miguel Estéfano Ave, 3687

São Paulo, SP 04301– 902

Brazil

H.H Chaminda Lakmal

Jeju National University

School of Marine Biomedical Sciences

Department of Marine Life Science

1 Ara 1-dong, 102 Jejudaehakno

Jeju 690-756

Republic of Korea

Pathum Chandika

Pukyong National University

Center for Marine-Integrated Biomedical Technology (BK21 Plus)

Department of Biomedical Engineering

Busan 608-737

Republic of Korea

Katarzyna Chojnacka

Wroclaw University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Department of Chemistry

ul. Smoluchowskiego 25

50-372 Wrocław

Poland

Maria G. Ciliberti

University of Foggia

Department of the Sciences of Agriculture

Food and Environment (SAFE)

Via Napoli, 25

71122 Foggia

Italy

Rosalia Contreras

Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE)

Molecular Microbiology Laboratory

Department of Marine Biotechnology

Carretera Ensenada-Tijuana No. 3918 Zona Playitas

Ensenada, BC

C.P. 22860

Mexico

Agnieszka Dębczak

New Chemical Syntheses Institute

Supercritical Extraction Department

Aleja Tysiąclecia Państwa Polskiego 13a

24-110 Puławy

Poland

Pradeep Dewapriya

Pukyong National University

Marine Biochemistry Laboratory

Department of Chemistry

Busan 608-737

Republic of Korea

Daniel A. Dias

Metabolomics Australia

The University of Melbourne

The School of Botany

Parkville

Victoria 3010

Australia

and

Deakin University, Centre for Chemistry and Biotechnology

School of Life and Environmental Science

Burwood

Victoria 3125

Australia

Agnieszka Dmytryk

Wrocław University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Department of Chemistry

ul. Smoluchowskiego 25

50-372 Wrocław

Poland

Zbigniew Dobrzañski

Wroclaw University of Environmental and Life Science

Department of Environment

Animal Hygiene and Animal Welfare

Chełmoñskiego 38C

51-631 Wrocław

Poland

Agnieszka Dobrzyñska-Inger

New Chemical Syntheses Institute

Supercritical Extraction Department

Aleja Tysiąclecia Państwa Polskiego 13a

24-110 Puławy

Poland

Jacinta S. D'Souza

UM-DAE Centre for Excellence in Basic Sciences

Department of Biology

Vidyanagari, UM Campus

Kalina, Santacruz (E)

Mumbai 400098

India

Joanna Fabrowska

Adam Mickiewicz University in Poznań

Department of Supramolecular Chemistry

Faculty of Chemistry

Umultowska 89b

61-614 Poznań

Poland

Julyana N. Farias

Post-Graduate Program in Biodiversidade Vegetal e Meio Ambiente

Instituto de Botânica

Miguel Estéfano Ave, 3687

São Paulo, SP 04301– 902

Brazil

Muhamad Firdaus

Brawijaya University

Department of Biochemistry

Faculty of Fisheries and Marine Sciences

Malang

East Java 65145

Indonesia

Mutue T. Fujii

Institute of Botany

Research Center in Phycology

Miguel Estéfano Ave, 3687

São Paulo, SP 04301– 902

Brazil

Henryk órecki

Wrocław University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Department of Chemistry

ul. Smoluchowskiego 25

50-372 Wrocław

Poland

Bogusława órka

Opole University

Department of Analytical and Ecological Chemistry

Faculty of Chemistry

Pl. Kopernika 11 a

45-040 Opole

Poland

Mateusz Gramza

Biotek Agriculture

Gać 64

55-200 Oława

Poland

Eduardo Morales Guerrero

Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE)

Department of Marine Biotechnology

Ensenada

BC 22860

Mexico

David R.A. Hill

The University of Melbourne

The Department of Chemical and Biomolecular Engineering

Parkville

Victoria 3010

Australia

You-Jin Jeon

Jeju National University

School of Marine Biomedical Sciences

Department of Marine Life Science

1 Ara 1-dong, 102 Jejudaehakno

Jeju 690-756

Republic of Korea

Won-Kyo Jung

Pukyong National University

Center for Marine-Integrated Biomedical Technology (BK21 Plus)

Department of Biomedical Engineering

Busan 608-737

Republic of Korea

and

Chosun University

Department of Marine Life Science

375 Seosuk-Dong

Dong-Gu

Gwangju 501-759

Republic of Korea

Fatih Karadeniz

Pukyong National University

Marine Bioprocess Research Center

599-1 Daeyeon 3-dong

Nam-gu

Busan 608-737

Republic of Korea

Mustafa Z. Karagozlu

Pukyong National University

Marine Bioprocess Research Center

599-1 Daeyeon 3-dong

Nam-gu

Busan 608-737

Republic of Korea

Keun Kim

The University of Suwon

Department of Bioscience and Biotechnology

Hwaseong-si 45-743

Republic of Korea

Se-Kwon Kim

Pukyong National University

Marine Bioprocess Research Center

Specialized Graduate School Science and Technology Convergence

Marine Biotechnology Laboratory

Department of Chemistry

Department of Marine-Bio Convergence Science

599-1 Daeyeon 3-dong, Nam-gu

Busan 608-737

Republic of Korea

and

Pukyong National University

Marine Biochemistry Laboratory Department of Chemistry

599-1 Daeyeon 3-dong, Nam-gu

Busan 608-737

Republic of Korea

Mariusz Korczyñski

Wroclaw University of Environmental and Life Science

Department of Environment

Animal Hygiene and Animal Welfare

Chełmoñskiego 38C

51-631 Wrocław

Poland

Dorota Kostrzewa

New Chemical Syntheses Institute

Supercritical Extraction Department

Aleja Tysiąclecia Państwa Polskiego 13a

24-110 Puławy

Poland

Sang-Hoon Lee

Korea Food Research Institute

Baekhyun-dong

Seongnam

Gyeonggi 463-746

Republic of Korea

and

University of Science and Technology

Pukyong National University Marine Bioprocess Research Center, Specialized

Daejeon 305-350

Republic of Korea

Bogusława Łę ska

Adam Mickiewicz University in Poznań

Department of Supramolecular Chemistry

Faculty of Chemistry

Umultowska 89b

61-614 Poznań

Poland

Yong Li

Changchun University of Chinese Medicine

Department of Pharmaceutical Sciences, 1035, Boshuo Road

Jing Yue Economic Development Zone

Chanchun City

Jilin Province

People's Republic of China

Yong-Xin Li

Marine Bioprocess Research Center

Pukyong National University

Busan 608-737

Republic of Korea

Jacek Lipok

Opole University

Department of Analytical and Ecological Chemistry

Faculty of Chemistry

Pl. Kopernika 11 a

45-040 Opole

Poland

Kuppusamy Manimaran

Annamalai University

Marine Biology, Centre of Advanced Study in Marine Biology

Faculty of Marine Science

Parangipettai 608 502

Tamil Nadu

India

Panchanathan Manivasagan

Pukyong National University

Marine Bioprocess Research Center

Department of Chemistry

Marine Biotechnology Laboratory

599-1 Daeyeon 3-dong

Nam-gu

Busan 608-737

Republic of Korea

Gregory J.O. Martin

The University of Melbourne

The Department of Chemical and Biomolecular Engineering

Parkville

Victoria 3010

Australia

Filipa Meireles

University of Coimbra IMAR-CMA

Department of Life Sciences

Faculty of Sciences and Technology

Rua da Matemática, nˆ 49

3001-455 Coimbra

Portugal

Beata Messyasz

Adam Mickiewicz University in Poznań

Department of Hydrobiology

Faculty of Biology

Umultowska 89

61-614 Poznań

Poland

Izabela Michalak

Wrocław University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Department of Chemistry

ul. Smoluchowskiego 25

50-372 Wrocław

Poland

Marcin Mikulewicz

Wroclaw Medical University

Department of Dentofacial Orthopedics and Orthodontics

ul. Krakowska 26

50-425 Wrocław

Poland

Rahmi Nurdiani

Brawijaya University

Department of Fishery Product Technology

Faculty of Fisheries and Marine Sciences

Malang

East Java 65145

Indonesia

Tatsuya Oda

Nagasaki University

Division of Biochemistry

Faculty of Fisheries

Bunkyo-machi 1-14

Nagasaki 852-8521

Japan

Jorge Olmos Soto

Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE)

Molecular Microbiology Laboratory

Department of Marine Biotechnology

Carretera Ensenada-Tijuana No. 3918

Ensenada, Baja California

C.P. 22860

Mexico

Ian L.D. Olmstead

The University of Melbourne

The Department of Chemical and Biomolecular Engineering

Parkville

Victoria 3010

Australia

Sebastian Opaliñski

Wroclaw University of Environmental and Life Science

Department of Environment

Animal Hygiene and Animal Welfare

Chełmoñskiego 38C

51-631 Wrocław

Poland

J. Paniagua-Michel

Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE)

Bioactive Compounds and Bioremediation Laboratory Department of Marine Biotechnology

Carretera Ensenada-Tijuana No. 3918 Zona Playitas

Ensenada

C.P. 22860

Mexico

Ratih Pangestuti

Research Center for Oceanography

Indonesian Institute of Sciences

Jl. Pasir Putih 1, Ancol Timur

Jakarta Utara 14430

Republic of Indonesia

Leonel Pereira

IMAR-CMA/MARE

Department of Life Sciences

Faculty of Sciences and Technology

University of Coimbra

Calçada Martim de Freitas

3000-456 Coimbra

Portugal

Marta Pikosz

Adam Mickiewicz University in Poznań

Department of Hydrobiology

Faculty of Biology

Umultowska 89

61-614 Poznań

Poland

Asep A. Prihanto

Brawijaya University

Department of Fishery Product Technology

Faculty of Fisheries and Marine Sciences

Malang

East Java 65145

Indonesia

Paulo J.A. Ribeiro-Claro

University of Aveiro

CICECO

Department of Chemistry

3810-193 Aveiro

Portugal

Pablo Riul

Universidade Federal da Paraíba

Departamento de Engenharia e Meio Ambiente, CCAE

58297-000 Rio Tinto, PB

Brazil

Edward Rój

New Chemical Syntheses Institute

Supercritical Extraction Department

Aleja Tysiąclecia Państwa Polskiego 13a

24-110 Puławy

Poland

Viviana P. Rubio

Universidad Autónoma de Baja California (UABC) Ensenada

Marine Science Faculty

Km 103 Carretera Tijuana-Ensenada

Ensenada, Baja California

C.P. 22860

México

Agnieszka Saeid

Wroclaw University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Faculty of Chemistry

ul. Smoluchowskiego 25

50-372 Wrocław

Poland

Kalpa W. Samarakoon

Jeju National University

School of Marine Biomedical Sciences

Department of Marine Life Science

1 Ara 1-dong, 102 Jejudaehakno

Jeju 690-756

Republic of Korea

Grzegorz Schroeder

Adam Mickiewicz University in Poznań

Department of Supramolecular Chemistry

Faculty of Chemistry

Umultowska 89b

61-614 Poznań

Poland

Kalimuthu Senthilkumar

Pukyong National University

Marine Bioprocess Research Center

Department of Chemistry

Marine Biotechnology Laboratory

Busan 608-737

Republic of Korea

Adam Słowiński}

Arysta LifeScience Poland

Przasnyska 6B

01-756 Warszawa

Poland

Katarzyna Stę pnik

Maria Curie-Skłodowska University

Department of Planar Chromatography

Faculty of Chemistry

Maria Curie-Skłodowska Square 3

20-031 Lublin

Poland

Marita Świniarska

Wroclaw University of Environmental and Life Science

Department of Environment

Animal Hygiene and Animal Welfare

Chełmoñskiego 38C

51-631 Wrocław

Poland

Łukasz Tuhy

Wrocław University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Department of Chemistry

ul. Smoluchowskiego 25

50-372 Wrocław

Poland

Mikinori Ueno

Nagasaki University

Division of Biochemistry

Faculty of Fisheries

Bunkyo-machi 1-14

Nagasaki 852-8521

Japan

Sirisha L. Vavilala

UM-DAE Centre for Excellence in Basic Sciences

Department of Biology

Vidyanagari, UM Campus, Kalina

Santacruz (E)

Mumbai 400098

India

Jayachandran Venkatesan

Pukyong National University

Marine Bioprocess Research Center

Department of Marine Bio Convergence Science

599-1 Daeyeon 3-dong

Busan 608-737

Republic of Korea

Piotr P. Wieczorek

Opole University

Department of Analytical and Ecological Chemistry

Faculty of Chemistry

Pl. Kopernika 11 a

45-040 Opole

Poland

Isuru Wijesekara

KU Leuven Toxicology and Pharmacology

Herestraat 49

Leuven 3000

Belgium

and

Pukyong National University

Department of Chemistry

Busan 608-737

Republic of Korea

Radoslaw Wilk

Wroclaw University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Department of Chemistry

ul. Smoluchowskiego street 25

50-372 Wrocław

Poland

Anna Witek-Krowiak

Wrocław University of Technology

Department of Chemistry

Division of Chemical Engineering

Norwida 4/6

50-373 Wrocław

Poland

Zuzanna Witkowska

Wroclaw University of Technology

Institute of Inorganic Technology and Mineral Fertilizers

Smoluchowskiego 25

50-373 Wrocław

Poland

Preface

Marine algae are popular food ingredients mainly in Asian countries such as Korea, Japan, and China. They are also well known for their health benefits because of the presence of bioactive components in them. Marine algae are rich in vitamins, minerals, dietary fibers, proteins, polysaccharides, and various functional polyphenols. Recently, several studies have demonstrated the variety of biological benefits associated with marine algal polyphenols, including antioxidant, anticoagulant, antibacterial, anti-inflammatory, and anticancer activities. These marine macroalgae have been classified based on pigmentation into brown (Phaeophyta), red (Rhodophyta), and green (Chlorophyta) types. Apart from food uses, including their main industrial use as thickeners and gelling agents, seaweeds are used widely as ingredients in nutraceutics and cosmetics and as fertilizers.

Marine Algae Extracts – Processes, Products, and Applications describes the characteristic features of marine algae cultivation, identification, production, process, and applications (biological, biomedical, food, and industrial). The book is divided into six parts: Part I provides the cultivation and identification processes of marine algae; Part II discusses the production and processing of marine algae; Part III provides an overview of the marine algae products; Part IV discusses the various biological applications of marine algae; Part V analyzes the numerous biomedical applications of marine algae; and Part VI examines the food and industrial applications of marine algae. Each part is a collection of comprehensive information on the past and present research on marine algae, compiled by proficient scientists worldwide. I personally intend to mention that the findings and the recent information provided in this book will be helpful to the upcoming researchers to establish a phenomenal investigation from a wide range of research areas.

I hope that the fundamental as well as applied contributions in this book serve as a potential research and development leads for the benefit of humankind. Altogether, marine algal biotechnology will be the hottest field in future toward the enrichment of targeted algal species, which further establishes a sustainable oceanic environment. This book would be a reference book for the emerging students in the academic and industrial research.

Busan, South Korea Se-Kwon Kim

10 Nov 2014

Acknowledgments

I would like to thank Wiley-Blackwell Publishers for their encouragement and suggestions to get this wonderful compilation published. I would also like to extend my sincere gratitude to all the contributors for providing help, support, and advice to accomplish this task. Further, I would like to thank Dr. Panchanathan Manivasagan and Dr. Jayachandran Venkatesan, who worked with me throughout the course of this book project. I strongly recommend this book for marine algae extracts researchers/students and hope that it helps to enhance their understanding in this field.

Se-Kwon Kim & Katarzyna Chojnacka

1

Introduction of Marine Algae Extracts

Katarzyna Chojnacka and Se-Kwon Kim

1.1 Introduction

Recently, there is increased interest in naturally produced active compounds as alternatives to synthetic substances. Although these compounds often show lower activity, they are nontoxic and do not leave residues. It has already been reflected by the projects of new law regulations in EU countries that have imposed legal restrictions on the use of xenobiotics as plant protection products or preservatives. In the European Union there are plans of new directives that impose additional environmental taxes, primarily because of the residues of active substances in the environment. This implies that there is a need to develop new and safe products of biological origin, with properties similar to the synthetic, in particular antimicrobial, antifungal, antioxidizing compounds, and colorants. These natural compounds are found in algal extracts (Table 1.1).

Table 1.1 Major compounds in algal extracts [2, 11, 19, 20]

Algal biomass have been used for centuries as food and medicine. The health promoting effects of algae were discovered as early as 1500 BC [1]. However, the biomass of algae gained interest as a source of chemicals and pharmaceuticals only recently. Nowadays, the production regime requires the use of extracts rather than the biomass itself, because of the formulation requirements (consistency, stability, color, flavor, etc.). Until now, algal products were available mainly as tablets, capsules, or liquid extracts, and sometimes were incorporated into food products, cosmetics, or products for plants [2]. In 2006, the market of microalgal biomass produced 5000 mg dry biomass/year and generated a turnover of 1.25 × 10⁹ USD [2]. The global sector of macroalgae is worth 6 billion USD, with main contribution from hydrocolloids and crop protection products [3]. Recently, compounds derived from algae (carotenoids, β-carotene astaxanthin, long-chain polyunsaturated fatty acids (PUFAs), docosahexaenoic acid) began to be produced on industrial scale [4]. Novel compounds isolated from algae possess a great further potential to be applied for their pharmacological and biological activity [4].

Seaweeds produce a vast spectrum of secondary metabolites because they live in nonfriendly environment but are not damaged photodynamically as they synthesize protective compounds and develop protecting mechanisms [5]. Environmental stress to which algae are exposed include rapid fluctuations of light intensity, temperature, osmotic stress, desiccation that lead to the formation of free radicals and oxidizing agents that lead to photodynamic damage [6].

1.2 Algal Biomass as a Useful Resource

Algae are the oldest photosynthetic organisms dating back to 3.8 billion years (prokaryotic cyanophytes) [7]. The number of species is estimated as 280 000 [7]. Algal biomass is being used as the raw material for different branches of industry and the global production is prevalently increasing [7]. Algae are photosynthetic organisms that convert light energy from the Sun into chemical energy stored in the form of chemical compounds in the process of photosynthesis [1]. A characteristic of algae is that they possess a simple reproductive structure [8]. The biomass of algae contains various compounds with diversified structures and functions that are synthesized in the response to stress conditions, for example, heat/cold, desiccation, salinity, osmotic stress, anaerobiosis, nitrogen deficiency, photooxidation, as protection from physiological stressors [1]. Algae are a diversified group of organisms and are divided into microalgae and macroalgae. The first group includes prokaryotic cyanobacteria and eukaryotic microalgae [9]. Algae are very diversified organisms when considering size (from unicellular microalgae to multicellular macroalgae) [10]. The basis for the classification of algae is pigmentation: green (Chlorophyceae), red (Rhodophyceae), and brown (Phaeophyceae) [11]. The difference concerns not only pigmentation, but also the type of storage material and the composition of cell wall polysaccharides [12]. Algae are simpler than terrestrial plants [12]. Algae could be considered as natural factories that produce bioactive compounds [13]. The composition of green algae: 10% protein, 35% carbohydrate, and 50% ash (Ca, Fe, P, Cl) [12].

Algae were in use since prehistory as the components of diet and as medicine [14]. Although the importance of algal industry is permanently increasing, there are some contradictions between Asian (Far East) and European ways of utilization of this resource [14]. In Europe, the biomass of seaweeds was treated as a sort of waste from seas and oceans [14]. Certainly, algal biomass is still an underutilized biological resource.

Algal biotechnology is divided into two branches: microalgal and macroalgal, with its unique specificity [15]. Microscopic algae are called microalgae; however, this term is not related to taxonomy. Among microalgae, cyanobacteria are distinguished, which are prokaryotic [15]. Macroalgal biotechnology includes the production of (phycocolloids agar-agar, alginates, carrageenan) from Rhodophyta and Phaeophyta, and the global value is 6 × 10⁹ per year [15]. At present, the main directions in macroalgal biotechnology are biofuels, agricultural biostimulants for crop plants, probiotics for aquaculture, soil bioremediation, wastewater treatment, and biomedical applications of extracted compounds (polyphenols, polysaccharides) [3]. Microalgal biotechnology refers to the production of different products: phycocyanin, carotenoids (β-carotene, astaxanthin), fatty acids and lipids, polysaccharides, immune modulators that find an application in health food, cosmetics, feed and food supplements, pharmaceuticals, and fuel production [15]. Microalgal groups of the major importance are cyanobacteria (Spirulina sp.), Chlorophyta (Chlorella sp., Dunaliella sp.), Rhodophyta (Porphyridium sp.), Bacillariophyta (Odontella sp., Phaeodactylum sp.) [15]. While macroalgae are harvested from natural habitats, microalgae are cultivated in artificial systems [15]. The products of microalgal biotechnology are coloring substances (astaxanthin, phycocyanin, phycoerythrin), antioxidants (β-carotene, tocopherol, antioxidant CO2 extract), and arachidonic acid (ARA), docosahexaenoic acid (DHA), and PUFA extracts [15].

1.3 Biologically Active Compounds Extracted from Algae

Because algae are coastal primary producers and have impressive possibilities to survive in extreme environmental conditions, in particular to trigger oxidative stress, they produce a variety of useful compounds [16]. Algae live in extreme conditions: fluctuating salinity, temperature, nutrients, and UV–vis irradiation [10]. Long periods of desiccation cause overproduction of reactive oxygen species, which is neutralized by physiological and biological mechanisms: the production of secondary metabolites [16]. Therefore, compounds isolated from the biomass of seaweeds possess biological activity. The biomass of algae contains many valuable components: minerals, vitamins (A, B, C, E), PUFAs (ω-3), amino acids, proteins, polysaccharides, lipids, and dietary fiber [17]. Many of these bioactive constituents can be extracted to obtain antioxidative, anti-inflammatory, antimicrobial, anticancer, antihypertensive products [11, 17]. Particularly useful are secondary metabolites with antiviral, antimalarial, anticancer properties [1]. Products derived from algae also contain polysaccharides, polyphenolic compounds, and terpenes [11]. Seaweeds and their extracts are added to food as antioxidants, antimicrobials, dietary fiber, and dietary iodine [6].

In various studies, strong antioxidative properties of compounds isolated from seaweeds were confirmed [18]. Antioxidative activity produces phlorotannins (polyphenolic compounds – 1–10% d.m. of brown seaweeds), alkaloids, terpenes, ascorbic acid, tocopherols, and carotenoids [18]. Antioxidants transform radicals into nonradicals by donating electrons and hydrogen, chelation of transition metals, and dissolving peroxidation compounds [6]. The role of antioxidants is to prevent lipid oxidation, inhibiting the formation of products as a result of oxidation, and consequently prolonging the shelf life of products [6]. Algae are a rich source of natural antioxidants and antimicrobial compounds [6].

The research on the composition of algal extracts concerns mainly antioxidants as an alternative to synthetic, because according to recent research these compounds if used as food additives are potential promoters of carcinogenesis [1]. The extracts modulate the oxidative stress and diseases related to radical scavenging effect: sesquiterpenoids and flavonoids (green alga Ulva lactuca), phlorotannins (brown alga Eisenia bicyclis, Ecklonia cava, E. kurome), phycobiliprotein, and phycocyanin (blue-green alga Spirulina platensis), which protect from DNA damage by H2O2 [17].

Anti-HIV – cyanovirin – protein from Nostoc ellipsosporum [1]

Photoprotective compounds – repair DNA damage – mycosporine-like amino acids, scytonemin enzymes (shock proteins) – superoxide dismutase, catalase, and peroxidase [1].

Microalgae contain carotenoids, PUFAs, phycobilins, sterols, polyhydroxyalkonates, and polysaccharides [9]. They can be considered as cosmeceuticals, nutraceuticals, and functional foods [9]. For instance, Spirulina contains lipids (6–13; 50% in the form of fatty acids), phycocyanin (20–28%), and carbohydrates (15–20%; mainly as polysaccharides) [21].

Algal cells contain phytochelatins – proteins synthesized in response to exposure to toxic metal ions [22]. However, the attempt to extract and use these proteins is not found in the available literature [22].

1.4 The Application of Products Derived from Algal Biomass

The global wild stocks of seaweeds yield 8 million mg of biomass [18]. In 2004, the contribution in the market was as follows: sea vegetables (88%), phycocolloids (11%), phycosupplements (1%), and the minor contribution of soil additives, agrochemicals, and animal feeds (totally, 6000 million USD) [14]. Algal extracts create a new market sector, because they can be used in a variety of products, for example, antioxidant capsules containing Spirulina extract, Chlorella extract in health drinks, oral capsules containing carotenoid extracts from Dunaliella [15]. Other examples of algal extracts-based products are pet functional food, biofertilizers (which increase water-binding capacity and serve as the source of minerals and substances promoting germination, growth of leaves and stems and flowering).

Of particular interest are antioxidants present in algae and their extracts, as the use of synthetic antioxidants has been restricted because of toxicity and health risks [23]. It is important to replace these synthetic compounds with natural antioxidants [23]. Antioxidative compounds from marine sources include various functional compounds, for example, tocopherols [19]. Lipid-soluble algal extracts can be used as protective functional ingredients [19]. Antioxidative properties of natural compounds from algae can prolong the shelf life of foods and cosmetics through delayed oxidation [11]. Natural anti-oxidants may also be useful in treating aging, UV-exposure, and diseases associated with oxidation [11]. Extracts from algae are used in cosmetics, for example, from Spirulina and Chlorella [2].

Polysaccharides isolated from algae are other important components of foods and cosmetics and in nutraceutical and pharmaceutical preparations and are produced mainly from seaweeds [21]. Polysaccharides (carrageenans, alginates) are used in food industry as edible packaging materials [6].

The main source of industrially exploited polysaccharides (alginate, agar, carrageenan) originates from the biomass of algae [12]. Algal biomass contains significantly higher levels of polysaccharides than terrestrial plants [12]. Algal polysaccharides differ from those in terrestrial plants: sulfate groups, additional sugar residues, high content of ionic groups, high solubility in water, and unique rheological properties [12].

Polysaccharide production includes the following steps: selection of raw material, stabilization and grinding of biomass, extraction and purification, precipitation, and drying [12].

1.4.1 Agriculture – For Plants

In modern agriculture, higher production should accompany lower environmental impact and higher sustainability [24]. These criteria fulfill biostimulants that improve efficiency of regular fertilization (increase the efficiency of nutrients uptake), enhance yield and the quality of crops, improve tolerance to environmental stress, and possess antioxidant properties [24]. Biostimulants are natural substances that promote growth, uptake of nutrients, and tolerance to abiotic stress and different climatic conditions [25]. Seaweed extracts can be used as foliar sprays for vegetables, grains, and flowers [24]. Plant growth regulators are defined as bioactive compounds. It is desired that they perform well and are degraded into products that are not harmful to the environment [26].

European Biostimulant Industry Council (EBIC) was established to help introduce agriculture biostimulants to the market and support regulatory EU authorities to describe biostimulants as innovative class of products, the production of which uses minimal synthetic processing. Biostimulants are approved in organic crops, with an important group of products derived from macroalgae [27].

Seaweeds have been used in the cultivation of plants since antiquity [28]. Seaweeds were composted since antiquity and used as soil amendments. The first industrial applications of seaweeds in agriculture were in 1944, as the new source of fiber [14]. At present, the extracts are applied directly to shoots foliarly or to soil [3]. The examples of algal extracts currently available on the market are Kelpak, Actiwave, and AlgaGreen [3]. Seaweed concentrates (e.g., Kelpak) are applied at low rates and have growth promoting effect following the presence of plant growth regulators (e.g., cytokinins and auxins, polyamines, putrescine, spermine) rather than nutrients [29]. These active substances increase the growth of nutrient-stressed plants [29].

In 1949, the product Maxicrop was developed [14]. Using liquid seaweed is advantageous, because plants respond immediately and positively (dilution 1 : 500); also, the ions of micronutrients (Cu, Co, Mn, Fe) are soluble at high pH and are chelated by partly hydrolyzed sulfated polysaccharides; soil crumb structure is improved (with alginate and fucoidan), microorganisms, root system, and plant growth are stimulated [14].

Extracts from seaweeds are useful in the cultivation of plants because they improve a wide range of physiological responses: increase crop yield, improve growth, improve plants' resistance to frost, serve as biofungicide and bioinsecticide, increase nutrients' uptake from soil because they contain plant growth regulators [30]. The extracts are used in low doses (high dilutions), because the active substances are efficient even in small quantities [30].

The compounds found in algal extracts that are important for plant growth are cytokinins, auxins, abscisic acid, vitamins, amino acids, and nutrients [24]. The outcome is the result of the synergistic effect of many compounds present in algal extracts [24]: phytohormones, betaines (organic osmolytes), polymers, nutrients, and alginic acid (soil conditioning agent that supports soil structure) [25, 28].

There are various reports of laboratory, pot, and field studies that aimed to test the plant growth stimulating properties of algal extracts. El-Baky et al. [31] investigated the effect of treatments with microalgae extracts (Spirulina maxima and Chlorella ellipsoida) on antioxidative properties in grains of wheat. The content of carotenoid, tocopherol, phenolic, and protein in grain was investigated. Antioxidant activity of ethanolic extracts showed the significant increase of radical scavenging activity in response to microalgal extracts treatment [31].

1.4.2 Functional Food

Functional food is defined as food that positively affects one or more physiological functions to increase the well-being and reduce the risk of suffering for diseases [8]. Recently, a new market for functional food has evolved, the food called food for the not-so-healthy [13]. Functional food is produced by the addition of active components. Functional food contains functional ingredients: micronutrients ω-3 fatty acids, linoleic acids, phytosterols, soluble fiber (inulin – prebiotics), probiotics, carotenoids, polyphenols, vitamins that present healthy effect on the organism [13]. New, biologically active natural ingredients (antioxidant, antiviral, antihypertensive) extracted from the biomass of algae are becoming important research objects in the area of food science and technology [10].

Algal extracts are the components of functional food, because they are considered as natural, biologically active components. The latter, beside nutrition, should have the beneficial influence on functions of the body by improving health or preventing from diseases [32]. Extracts from Spirulina can be added to functional foods because of antioxidant, antimicrobial, anti-inflammatory, antiviral, and antitumoral properties of the compounds (phycocyanins, carotenoids, phenolic acids, and ω-3 and six PUFAs) [32].

Algae are used as dietary supplements that are classified into three groups: (i) Spirulina platensis, (ii) Aph. flos-aquae, and (iii) Chlorella pyrenoidosa [33]. The biomass of these microalgae is obtained either from lakes or by cultivation in artificial ponds [33]. Algae can be cultivated, in which the growth rate is high and in some cases there is a possibility of controlling the production of active compounds by adjusting cultivation conditions [10].

The potential use of brown seaweed extracts to inhibit the growth of microorganisms responsible for food spoilage and pathogenic microorganisms was also investigated [5]. The addition of 6% of the extract substantially reduced the growth of nondesired microflora [5].

1.4.3 Cosmetics

Microalgae, the biomass of which is to be used as the raw material for isolation of beneficial compounds, are cultivated in artificial systems that provide the biomass that is free of impurities [7]. Algal extracts are useful in the skin care market as well because they support regeneration of tissues and reduce wrinkles, in particular, the extracts from Spirulina (which repair signs of aging, prevent stria formation) and Chlorella (stimulate collagen synthesis) [2]. The properties of microalgal extracts include reduction of intracellular oxidative stress and synthesis of collagen [7].

Extracts from the following microalgae are produced commercially for cosmetic industry [7]:

Nannochloropsis oculata – vitamin B12, vitamin C, and antioxidants

Dunaliella salina – pigment industry (carotenes), amino acids, and polyphenols

Chlorella vulgaris – proteins, and inorganics substances.

1.4.4 Pharmaceuticals

Algal extracts can replace commercial antibiotics in disease treatments [34]. Biologically active metabolites isolated from marine algae have the potential to be used as pharmaceuticals because they inhibit the growth of bacteria, viruses, and fungi [34]. The chemicals are macrolides, cyclic peptides, proteins, polyketides, sesquiterpenes, terpenes, and fatty acids [34]. Cavallo et al. [34] investigated the effect of lipid extracts from six algae and their antibacterial activity against fish pathogens and found that they can be used as antibacterial, health promoting feed for aquaculture.

Extracts from Spirulina are active against viruses (herpes, influenza, cytomegalovirus) and inhibit carcinogenesis [35]. Spirulina is the source of vitamin A that is highly absorbable [36].

Hot water extract from Spirulina supports human immune system by the improvement of immune markers in blood (higher level of gamma interferon and interleukin-12p40 and toll-like receptors) and acts directly on myeloid lineages and natural killer-cells (NK cells) [35]. Immulina is a polysaccharide found in the extract from Spirulina that activates monocytes. Water extracts also showed antiviral activity [35].

1.4.5 Fuels

Seaweed extracts can be the resource to produce liquid fuels (ethanol), because of high carbohydrates (laminaran, mannitol) content [37]. Seaweeds can be bioconverted to methane [37].

1.4.6 Antifouling Compounds

Extracts from marine algae (e.g., Enteromorpha prolifera) contain compounds that have antifouling properties toward, for example, mussels (Mytilus edulis) and larval settlement: tannins (Sargassum natans), bromophenol (Rhodomela larix), diterpenes (Dictyota menstrualis), and halogenated furanones (Delisea pulchra). These compounds have the potential in the prevention from fouling of ship hulls and aquaculture nets instead of organotin or paints based on toxic metals [38].

1.5 Extraction Technology

Seaweed industry was established in 1950s [3]. The production concerned mainly low-cost fertilizers and food [3]. For the first time liquefaction of seaweeds was undertaken in 1857 by compressing [28]. The goal was to obtain the formulation that is transportable over long distances [28]. Algal extracts were obtained and patented in 1952 by alkaline extraction [3]. Another process was milling in low temperature [28].

Although natural extracts possess a great applicable potential, the problem with natural products is variable composition of extracts because of fluctuations in the raw material (season, location), different extraction techniques [12]. Extraction methods vary and the following can be distinguished: ethanol, methanol, enzymatic [17], composting, supercritical CO2 extraction with cosolvents.

In the elaboration of a new extraction technology, it is necessary to select the target bioactive compound, select the species of alga for extraction containing the compound of interest, select the operation conditions to find a compromise between the yield and purity, and consider if large enough resources of the algae are available.

It is essential to develop appropriate, quick, cost-efficient, and environmentally friendly methods of extraction that aim to isolate biologically active compounds of interest [10] without loss of their activity. It is essential to develop extraction procedures that involve the use of specific solvents and processes [8].

The production of algal extracts consists of several unit operations [7]:

Upstream processing – preparation for cultivation

Cultivation – in photobioreactors

Downstream processing – cell harvesting, rehydration and hot water extraction, centrifugation, and ultrafiltration

Formulation, preservation, and conditioning.

Traditional extraction techniques (soxhlet) solid–liquid extraction (SLE), liquid–liquid extraction (LLE) consume large quantities of solvents and require high extraction times [8]. These procedures present low yield of extraction and low selectivity toward bioactive compounds [8]. Because of the lack of automation, reproducibility is low [8]. Recently developed techniques supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), accelerated solvent extraction (ASE), pressurized hot water extraction (PHWE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) have further reduced these limitations [8]:

Solvent extraction – large quantities of toxic organic solvents are used, long time of extraction, laborious, low selectivity, low extraction yields, and not mild conditions (temperature, light, oxygen) [32].

Pressure liquid extraction – less solvent, shorter time of extraction, automated, no oxygen, and no light [32].

Supercritical fluid extraction – technique used to isolate active components from natural materials [32].

FE uses solvents at temperatures and pressures above their critical point and is used to extract compounds from biomasses [8]. In this technique, the consumption of toxic organic solvents is reduced and the main solvent used is CO2 [8]. The disadvantage is low polarity of CO2 and resulting necessity of the use of polar modifiers or cosolvents [8]. Advantages are high diffusivity, easiness in the control of temperature and pressure (possibility of modification of solvent strength), and obtaining solvent-free extracts [8].

Extraction of biologically active compounds from algal biomass is not selective. The extract is a mixture of different compounds [11]. The factors that influence the composition and thus the activity of algal extracts depend on species, environmental conditions, season of the year, age, geographical location, and processing technologies [11]. For instance, ethanol was found to be more efficient in the extraction of polyphenols than water [23]. Seaweed extracts contain PUFAs (in particular ω-3 long chain PUFA) that have several health promoting effects and have the potential to be useful in treatment or reducing symptoms of: cardiovascular disease, depression, rheumatoid arthritis, and cancer [19].

Chaiklahan et al. [21] optimized the extraction of polysaccharides from Spirulina sp. It was found that the mostly significant operation conditions were temperature and solid to liquid ratio and time. The extract contains rhamnose and phenolic content [21].

Seaweed concentrates are used as supplementary soil conditioners that promote plant growth and improve crop yield [29]. An example product is Kelpak® from Ecklonia maxima [29]. These products are used in very low doses and contain, for example, cytokinins and auxins that are plant growth regulators [29]. Seaweed extracts are particularly useful if applied on plants that are nutrient-stressed [29].

1.6 Conclusions

Algae are a useful raw material for biobased economy, because their cells contain a vast array of useful compounds with high biological activity. Biomass of algae is certainly an underestimated resource. In the process of extraction it is possible to draw the valuable compounds closed in the algal cells. However, this should be carried out in such a way that the structure and thus the properties of the compounds are not destroyed and that the solvent used does not limit their use as safe components of products for plants, animals, and human.

There are many ways to implement the extraction process and this is thoroughly discussed in this book. In addition to developing extraction technology, it is very important to assess the utilitarian values of the extracts, which can be documented in application studies of extracts in real systems.

Preparation of algal extracts represents a new approach in the preparation of natural products with a standardized composition, as compared with the biomass itself and certainly will be a future for algal industry.

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Part I

Cultivation and Identification of Marine Algae

2

Identification and Ecology of Macroalgae Species Existing in Poland

Beata Messyasz, Marta Pikosz, Grzegorz Schroeder, Bogusława Łęska and Joanna Fabrowska

2.1 Introduction

Algae are most common organisms in aquatic environment and a very diverse group in terms of ecological, taxonomic, morphological, and biochemical aspects [1–5]. Microscopic algae float freely in water and form plankton, which plays an important role in maintaining the balance of the aquatic habitat [6]. Macroscopic algae exhibit complex degrees of organization of thalli. Their main representatives are marine red algae (Rhodophyta), brown algae (Phaeophyta), and green algae (Chlorophyta), whose names are derived from the characteristic pigments phycoerythrin, fucoxanthin, and chlorophyll, respectively. The thalli of these algae, depending on the species, can reach a size of a few microns up to several meters. In the case of large marine thalli leaf-like (phylloid), stem-like (cauloid), and roots-like (rhizoids) forms can be found [6, 7].

On the basis of a wide morphological diversity and biochemical characteristics algae have traditionally been classified into several taxonomic groups (phyla). Organizing algae according to the principles of the phylogenetic system is still rather difficult. The first system of algae classification based on the theory of parallel development of monophyletic groups of algae was derived from flagellates and then included various degrees of morphological organization. According to this compilation, highly organized filamentous and pseudoparenchymatous forms arose from primitive unicellular flagellate algal cells [2, 3, 7–9]. In this system, representatives of marine and freshwater macroalgae are included, such as green algae (e.g., Ulva, Cladophora), red algae (e.g., Batrachospermum, Porphyra, Polysiphonia, Furcellaria), brown algae (e.g., Fucus, Laminaria), cyanobacteria (Tolypothrix, Scytonema, Nostoc), and xanthophyceans (e.g., Vaucheria, Tribonema) [10, 11]. Some of these genera are found in freshwater ecosystems in abundant quantities (Figure 2.1). Interestingly, often their presence in the aquatic reservoirs is generally defined as filamentous green algae by the researchers without identifying the species structure of such mats. Significantly, this makes it difficult to characterize the ecology of individual species, and a comparison with neighboring countries or regions is also not possible as it will require a thorough verification of such incompletely reported occurrences.

nfg001

Figure 2.1 Systematic diagram of most often recorded filamentous algae from water ecosystems in Poland.

In the marine environment, in contrast to the freshwater, the occurrence of macroscopic algae is influenced by the availability of light. Zonation takes place, where red algae can develop in the lowest part of the water column. Likewise, mass macroalgae developments are also found in inland water source; however, the scale of such blooms is lower than it is in marine water because of the smaller size of the reservoirs. These are mainly representatives of green algae and to a lesser extent of xanthophyceans (Figure 2.2). Cyanobacteria, irrespective of the type of water ecosystem, are only an accompanied group in the macroalgal associations as they do not create a large surface mat by themselves. Their filamentous forms can grow as a thin mat over stones, break away and become free-floating (Stigonema), form dark tufted mats (Scytonema), or tangle among submerged vegetation (Tolypothrix). On the contrary, red and brown algae are predominantly marine. Only few species are found in freshwaters (e.g., Batrachospermum, Lemanea). There are many others that have not been fully studied and their ecological characteristics are still not well described.

nfgz002

Figure 2.2 Massive development of filamentous green algae forming mats from Wielkopolska region (Poland): (a) Ulva intestinalis in Nielba river (Photo by M. Koperski.); (b) Oedogonium in the Konojad pond; (c) filaments of Cladophora glomerata in Lake Durowskie; (d, e) Cladophora glomerata in Lake Oporzyn; (f) Cladophora fracta in the Malta Reservoir; (g) long filaments of Cladophora glomerata in the Mogilnica river; (h) Cladophora rivularis in the Konojad pond; and (i) Zygnemataceae in the artificial pond in Poznań (Photo by J. Rosińska).

Macroscopic green algae and xanthophyceans although usually free-floating thalli forming dark green patches may be attached when they are young and before breaking free. The speed of the growth of macroscopic algae biomass is influenced by environmental conditions that vary according to the season. These algae in terms of longevity are the annual forms. Therefore, the variability of macrogreen algae mats will concern the composition and species diversity, the structure of patches (loose or dense) as well as the occupied area. However, in each case the rapid algal growth permits their rapid settling on the available substrates. Our long-term studies have shown that successional stages are less predictable in freshwater ecosystems than in marine ones although one clearly can distinguish the spring phase with filamentous ephemerals such as Ulothrix or Tribonema and the summer phase with a dense carpet/mats of Cladophora, Oedogonium, or Ulva [12].

2.2 Collection of Macroalgal Thalli and Culture Conditions

Macroalgae collection is dependent on the habitat in which these organisms occur. Because of the considerable depth of water where these are available a collector often needs a boat. For the habitat characterization, the basic physiochemical parameters of the water (temperature, conduction, concentration of oxygen and Cl− as well as the pH) at the sites of macroalgae thalli are measured with the use of the YSI Professional Plus hand-held multiparameter meter. Thalli samples are collected manually from the middle of the mat, which is formed by the macroalgae at the sampling site. When a macroalgal mat is not floating on the water surface, thalli samples are collected under water, often by gathering individual creepings at the bottom or tangled in aquatic vegetation. It is recommended that about 500 g of algal thalli is collected, which are rinsed five times with water from a given site. The thalli are put in a plastic container and transported in a fridge (at 4 °C) to the laboratory, where they are rinsed again a couple of times with distilled water in order to remove any algae, vascular plants (lemnids), sand, or snails stuck to them. Next, the purified thalli belonging to one genus or species are divided into smaller portions: (i) 10 g is used for the microscopic analysis and morphometric measurements of both thalli and cells, (ii) 20 g is used to perform the herbarium specimens, (iii) another 30 g of the sample is preserved (including 10 g of the material frozen in the temperature of −10 °C, 10 g preserved with 4% formalin solution, and 10 g preserved with Lugol's solution; put into 100 ml plastic containers), and (iv) to analyze the chemical composition, 20 g of thalli is dried for 30 min on a cellulose filter paper at room temperature and then for 2 h at 105 °C. The obtained dry mass is stored in 100 ml plastic containers. The remaining 400 g of the collected sample is placed in an 10 l aquarium with water filtered from the habitat or the Wang medium and next deposited into phytotrons (at 250 µmol photons m−2 sek−1, period 12 : 12, temperature 21 °C) to conduct macrocultures in open or closed systems [13].

In order to obtain high quality raw materials for the production of food products and cosmetics, cultures of algae are treated more frequently under specially defined conditions to increase the biomass production. Open cultures are mainly related to algae culture on a large scale or in cases where experimental sets occupy a large surface. The main element of such a culture is a container with water for the growth of algae. Other components are subject to various modifications, depending on the needs and purpose of the experiment. Cultures focused on obtaining the highest algal biomass are built from containers of large capacity and equipped only with monitoring devices and aerator [14]. Sets for breeding algae with other organisms (snails, shrimps) are more complicated. The latter are chosen to examine the mutual ecological relations [15, 16]. Such sets consist of several smaller containers connected by canals with water circulation. More complex sets of open culture are equipped with pipes supplying water enriched with nutrients, the heater controlling the water temperature, devices for simulating the movements of water or artificial light sources [17]. And, the thalli of algae (instead of freely floating on the water) are deposited on the special nets or other supports [18].

Phytotron chambers are used, which permit the cultivation of algae under certain simulated conditions. In these chambers several environmental factors can be modeled, such as (i) air temperature (set by the heating and cooling systems, maintaining the temperature regardless of the surroundings), (ii) circulation and humidity (provided by a system of fans and filters), and (iii) intensity and color of light (system of lamps and day and night cycle). Embedded microprocessors allow the automatization of processes and controlling proper operational parameters.

Tunable components of the culture medium are nitrogen, phosphorus, pH buffers, salinity (minimum 30 ppt), and optionally trace metals and vitamins as defined by the medium recipe. In the case of marine algae culture, to obtain the required amount of biomass, the physicochemical properties of the marine water from the site from which a specific macroscopic algae species are harvested are analyzed; then an appropriate amount of chemicals is added , which leads to certain nutrient and trace element concentrations. A key factor that determines the success of the cultured freshwater forms of macroalgae is to select the appropriate media. On the basis of the observations of the concentration of nutrients for the macroalgae, it has been found that Wang's culture medium or the Benecke medium (with some modifications) [13] are most suitable. The relatively high contents of N and P present in these media are similar to those existing in a eutrophic reservoir habitat – preferred by mat-forming algae. NaCl is added to the medium, for example, in the case of the culture of freshwater forms of Ulva (preferences are different for individual species) to complete chloride ions or the addition of trace elements mixture in the cultivation of other filamentous green algae species. A very important aspect of culture preparation is the identification of the species that were collected for testing. Species of the genus Cladophora, Ulva differ substantially in terms of levels of certain nutrient preference. It is therefore necessary to adjust the amount of the nutrient element in the medium to the requirements of the identified macroalgae.

2.3 Macroalgae Forming a Large Biomass in Inland Waters of Poland

This chapter relates to macroalgae living in the freshwaters of Poland. However, some of these algae such as Ulva spp. (Enteromorpha spp.), Cladophora spp., and Vaucheria spp. are represented also in the marine waters of the Polish coast where they cover the stone bottom. In some places stoneworts are also rooted (Chara spp.). On the contrary, brown alga Fucus vesiculosus L., which is specified as the most characteristic plant of the Baltic Sea, has become extinct on the Polish coast completely [10, 11, 19, 20]. Thirty years ago, it occurred abundantly at the stony bottom near the cliffs and in the Puck Bay together with the aquatic plant Zostera marina L. and red alga Furcellaria lumbricalis (Hud.) Lam., creating an association of underwater meadows with an incredible biodiversity. Nowadays, fragments of this brown alga are more often found on our beaches as detached by water from the other parts of the Baltic Sea. A very common filamentous brown alga in the southern Baltic now is Pilayella littoralis (L.) Kjell. [11, 21]. Its delicate and thin thalli are strongly and diversely branched. It creates dense bushes of yellow brown color reaching several centimeters in length. During the summer it grows strongly. Moreover, this species has a tendency to spread rapidly along the Polish coast.

In the case of tubular forms of marine green algae from the genus Ulva (Enteromorpha), Ulva compressa L. and Ulva plumosa Hud. are present widely while less represented (and in isolated locations) are Ulva clathrata (Roth) Ag., Ulva linza L., Ulva prolifera O.F. Müller, and Ulva torta (Mert.) Trev. [22–25]. The pale green thallus of U. compressa is shaped like a flattened tube with delicate and very thin cell walls. Length of thalli can reach dozens of centimeters and grow up to 2 cm in width. They are often distended in the form of bubbles because of the air penetrating the thalli. Its thallus narrows in intervals, from which new branches extend. This green alga strongly attaches via disc-like basal cell to solid substrates. Waves do not cut these, but only sways them. However, U. plumosa, which is very common in the Baltic Sea, is heavily branched and forms a bundle of long, delicate thalli in an intensive and luscious green color. In the case of a soft bottom it is attached to shells or pebbles. Its filamentous thalli reach more than 30 cm in length.

In addition, in marine ecosystems, despite the most common filamentous ones like Cladophora glomerata (L.) Kütz., there are also other species, such as Cladophora fracta (O.F. Müller ex Vahl) Kütz. (Figure 2.2f), Cladophora albida (Nees) Kütz., Cladophora sericea (Hud.) Kütz. [11, 22, 23, 26]. A very interesting species is Cladophora rupestris (L.) Kütz., which has a characteristically branched thallus. From the apical part of the filament protrude a few branches, from each of them arise again three to four branches forming a kind of brushes. It is easy to identify because of the strong ramification thalli of this alga and its rigidity. Such light green and fluffy bushes can reach about 15 cm in height. It tolerates a wide range of temperatures and thus is a perennial species. However, it does not grow during winter and is not fruitful.

On the basis of the findings from our long-term studies and all available literature, the characteristics of the biology and ecology of select macroalgae taxa are described below. For each species the same pattern of presentation is chosen, including macro- and microscopic appearance, habitat preferences, place of occurrence, and characteristics of the communities in which they were recorded.

Synonyms: No data.

Common names: wstężnica (Polish) [27].

Macroscopic appearance: Unbranched thin filaments.

Microscopic appearance: Cylindrical cells 4.5–7 µm thick, 0.5–1.5 times as long as broad, 1 pyrenoid [28]. Cells cylindrical, 8–14 µm long, 6–9 µm wide, end of cells rounded, single pyrenoid, containing numerous starch grains and central nucleus [29]. Cylindrical cells 4.5–7 µm width and 0.5–1.5 times longer [27]. Square shape cells (width: 2.5–6 µm length: 5–6 µm) with two chloroplast located on the sidewall, rounded apical cell (own research).

Habitat: Reported as a terrestrial species, it may be attached or free-floating on the water. The alga has a cosmopolitan distribution mainly in stagnant, flowing waters particularly at cooler times of the year. Light (grassy) green, forming delicate watt. It can also be attached to submerged stones or wood.

Communities: In the littoral zone Ulothrix forming mats with Oedogonium, Spirogyra, Zygnema, and Mougeotia [30]. Often included in phytoplankton community, were noted with Tribonema aequale and T. vulgare [12].

Distribution: This species is found in a variety of small pools and shallow water bodies as well as in soil but in small amounts. Massive occurrence was noted in April in midfield pond in Konojad village [12]. Ulothrix species were noted on the Spitsbergen in terrestrial ecosystems [31].

Remarks: About 30 species of Ulothrix genera are known.

ULVA FLEXUOSA SUBSP. PILIFERA (Kütz.) Bliding 1963 (Figure 2.3i,j) (Chlorophyta, Ulvophyceae).

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Figure 2.3 Morphology of (a) highly branched thalli of Cladophora glomerata; (b) numbers of nucleus in C. glomerata cell after acetocarmine staining; (c) branched Cladophora rivularis; (d) ball form of Aegagropila linnaei; (e) filament of A. linnaei with characteristic opposite and subterminal branches; (f) unbranched filament of Rhizoclonium sp.; (g) filament with H-shape cell membrane of Microspora sp.; (h) Ulva intestinalis thallus with proliferation in the lower part; (i) thalli of Ulva flexuosa subsp. pilifera; (j) cells of Ulva sp.; (k) unbranched filament of Oedogonium capillare with pyrenoids; (l) Oedogonium sp. with apical cell; (m) Ulothrix variabilis with single, parietal, girdle-shaped chloroplast; (n) coenocystic, hollow tube of Vaucheria sp.; (o) antheridium of Vaucheria sp.; (p) Tribonema aequale with H-shape pieces; and (q) filaments of Tribonema and Ulothrix.

Synonym: Enteromorpha pilifera Kützing.

Common names: błonica oszczepowata, watka oszczepowata (Polish) [32].

Macroscopic appearance: Monostromatic tubular thalli long, 15–30 × 1–3 cm [33] and according to Rybak and Messyasz [32] 15–41 × 0.4–4.2 cm. Thalli of macroalgae can reach length up to 1 m [27]. Thalli with little proliferation