Innovative bio-products for agriculture : algal extracts in products for humans, animals and plants 9781634855587, 1634855582, 9781634855723, 1634855728

Table of contents :
Content: Preface
Innovative Technology of Algal Extracts Obtained by Supercritical Fluid Extraction Useful in the Products for Plants, Animals & Human
Extraction Studies of Spirulina Platensis using ASE & SFE Methods
The Analysis of Technical & Economic (TEA) Aspects of Natural Raw Materials Extraction with Supercritical Carbon Dioxide
Identification of Biologically Active Compounds & Assessment of Commercial Properties of Algal Extracts as Cosmetic Ingredients
Biologically Active Compounds in Algae & Their Application in Plant Growth Stimulation
The Algae Biomass in Animal Production
Index.

Citation preview

BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

INNOVATIVE BIO-PRODUCTS FOR AGRICULTURE ALGAL EXTRACTS IN PRODUCTS FOR HUMANS, ANIMALS AND PLANTS

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

INNOVATIVE BIO-PRODUCTS FOR AGRICULTURE ALGAL EXTRACTS IN PRODUCTS FOR HUMANS, ANIMALS AND PLANTS KATARZYNA CHOJNACKA AND IZABELA MICHALAK EDITORS

New York

Copyright © 2016 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470

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Library of Congress Cataloging-in-Publication Data Names: Chojnacka, Katarzyna, editor. | Michalak, Izabela, editor. Title: Innovative bio-products for agriculture : algal extracts in products for humans, animals and plants / editors, Katarzyna Chojnacka and Izabela Michalak (Faculty of Chemistry, Wroclaw University of Technology, Poland). Description: Hauppauge, New York : Nova Science Publisher's, Inc., [2016] | Series: Biotechnology in agriculture, industry and medicine | Includes bibliographical references and index. Identifiers: LCCN 2016024101 (print) | LCCN 2016031358 (ebook) | ISBN 9781634855587 (hardcover) | ISBN 9781634855723 (ebook) | ISBN 9781634855723 () Subjects: LCSH: Marine algae--Utilization. Classification: LCC QK567 .I56 2016 (print) | LCC QK567 (ebook) | DDC 579.8/177--dc23 LC record available at https://lccn.loc.gov/2016024101

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

vii Innovative Technology of Algal Extracts Obtained by Supercritical Fluid Extraction Useful in the Products for Plants, Animals and Human Izabela Michalak and Katarzyna Chojnacka Extraction Studies of Spirulina platensis Using ASE and SFE Methods Agnieszka Dobrzyńska-Inger, Dorota Kostrzewa and Edward Rój

1

29

The Analysis of Technical and Economic (TEA) Aspects of Natural Raw Materials Extraction with Supercritical Carbon Dioxide Kazimierz Kozłowski, Edward Rój, Agnieszka Dobrzyńska-Inger and Dorota Kostrzewa

43

Identification of Biologically Active Compounds and Assessment of Commercial Properties of Algal Extracts as Cosmetic Ingredients Grzegorz Schroeder, Beata Messyasz and Bogusława Łęska

73

Biologically Active Compounds in Algae and Their Application in Plant Growth Stimulation Bogusława Górka, Karolina Kucab, Jacek Lipok and Piotr P Wieczorek

101

vi Chapter 6

Contents The Algae Biomass in Animal Production Marita Świniarska, Mariusz Korczyński, Sebastian Opaliński, Zbigniew Dobrzański, Anita Baron, Maria Chrzanowska and Łukasz Bobak

129

Editors' Contact Information

155

Index

157

PREFACE The aim of this book is to highlight the utilization of algae for the development of useful products using environmentally friendly technologies such as supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE). Each chapter is a collection of comprehensive information on the present research on algae in different applications. The primary emphasis of this book however, is concerned with the products used for agriculture. Algal extracts were presented in this book also as a biologically active component that adds value to products for humans (cosmetics), animals (pro-health supplements) and plants (plant growth biostimulants). Chapter 1 - In the present chapter, supercritical fluid extraction of seaweed collected from the Baltic Sea (Polysiphonia, Ulva and Cladophora) and microalga Spirulina (Arthrospira) was described. This novel extraction technique was discussed in terms of the isolation from the algal biomass of antioxidants (e.g., polyphenols), lipids (including polyunsaturated fatty acids), elements (micro-, macro- and toxic elelemnts), as well as plant growth hormones. On the basis of the composition of the obtained extract, their potential applications were proposed. Supercritical algal extracts can constitute a component of the products for plants (biostimulants of plant growth), animals (feed additives) and for human (cosmetics, diet supplement, pharmaceuticals, nutraceuticals). This wide application is possible due to the character of the extract – it is free of the organic solvents, which are commonly used in the clasical solvent extrcation. Chapter 2 - Spirulina platensis was extracted using supercritical fluid extraction and accelerated solvent extraction. The two extraction techniques were compared using different solvents. SFE was performed in the laboratory scale using carbon dioxide as solvent. The response surface methodology

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(RSM) was applied to evaluate the effect of three independent variables: temperature (40-60°C), pressure (35-65 MPa) and extraction time (45-135 min) on the total extraction yield. The relationship between the yield and the process variables was determined by a second order poly-nominal equation. Comparing the yields of the three extraction methods it was established that the highest yield value was reached by ASE with acetone, but the extraction yield with supercritical carbon dioxide was higher than yield by ASE with hexane. Chapter 3 - It is a well-known fact that the primary objective of any business and production activity of every entrepreneurship is effective management which covers its current needs and ensures constant development. Regular technical-economic analyses (TEA) of current and forecast activity with a special regard to the changing conditions and TEA of new undertakings referring to new products, services and investments are necessary to ensure effective management. Only simplified methods of specifying technical cost of producing some of the extracts and optimizing some of extract parameters can be found in the available literature, however, there is no complex presentation of a simple method of carrying out TEA. This paper presents the method of carrying out TEA, which is a basic method of the evaluation of economic efficiency regarding the production of a well-known or new extract, processing a well-known or new raw material in current of forecast conditions and in the existing of new plant, taking into account:  

the level and technical structure of manufacturing the product and/or processing the raw material, the specification of influence of particular parameters on the level and technical structure of the cost of manufacturing the product and processing the raw material.

The result of TEA may be one of the major criteria to be used for the cost estimation of extraction process in the existing plant and for taking up a decision regarding the advisability of the construction of a new plant and its size. The other objective of TEA is to specify and take into account all the cost factors connected with extraction process, which prevents accidental mistakes and contributes to lower extraction costs. Chapter 4 - From among the variety of materials used as cosmetic ingredients, algae have become increasingly popular. Extracts from a single species of freshwater macroalgae ensure higher reproducibility of biologically

Preface

ix

active substances than the extract from mixtures of marine macroalgae, which each time vary in species composition. The composition and quantity of organic compounds derived from freshwater macroalgae (mostly from Cladophora glomerata (L.) Kütz.) depends mainly on: the quality of raw materials, the degree of fragmentation of the algal biomass, the method and time of extraction and preservation and protection of extract. The diversity of effects of algae extracts have stimulated their use in different types of cosmetics such as face creams, body lotions, make-up, lotions, toners, masks, cosmetics ultraviolet filters, bath products, hair care preparations, anti- type aging, moisturizing, anti-acne, anti-inflammatory, anti-cellulite, anti-bacterial and others. Macroalgae can be used as an additive to cosmetics in micronized form or as extracts. Prior to application cosmetics must be subjected to mandatory tests and optionally to additional tests. The mandatory tests include dermatological tests, application, durability test and safety assessment. According to the conditions of testing the test methods are also divided into in vitro, ex vivo and in vivo ones. The in vitro assays are performed at the initial phase of testing of cosmetic products and include studies of biological processes in artificial conditions, outside living organisms. The ex vivo tests rely on the assessment of the performance of components and finished cosmetics on human skin in laboratory. The most important studies before the appearance a new cosmetic product on the market are application tests (in vivo) on human skin. They allow determination of the activity of the product in the most effective and reliable way. Chapter 5 - In recent years the bioactive substances of natural origin gained more and more interest. Among such substances, the compounds isolated from microalgae and seaweeds, mainly the polyphenols, polysaccharides, plant hormones, pigments and fatty acids, an find an application in many industries, particularly in medicine, pharmaceutics, cosmetics and agriculture. Biostimulants from seaweeds have been used and manufactured for years, giving confirmation of its effectiveness in field trials, enhancing the growth of crops giving better yields. Nowadays, the legislation processes are more difficult and intricate, so it is harder to register new products. Everyone put more attention on the answers for the questions like: how does it really work, what is the actual active component, is it single substance or a group of them, what is the concentration of these compounds in the seaweed extract. So, basically the scientific proof is needed in order to verify the final product and fulfill the requirements in law regulations. Strongly developed these days

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fields of knowledge instrumental analysis and analytical chemistry will satisfy the curiosity and come back with the answer for all these questions. That is why, the analytical methods used for the determination of the active substances present in seaweeds, as well as the potential of these compounds in application, mainly as useful products stimulating directly and indirectly the plant growth in agriculture, are described in this chapter. Chapter 6 - Recently, a need to search for innovative products containing naturally derived, animal-safe ingredients has been observed. Algae are a valuable source of useful natural ingredients that exhibit beneficial activities such as, antioxidative, antimicrobial, antiviral and health-improving. Seaweeds, which contain polysaccharides, essential amino acids, pigments, polyphenols and unsaturated fatty acids, have favorable effects on farm animal health status and may be used as a feed supplement in order to enrich animal origin products. The possibility of applying microalgae, microalgae extract and postextraction residue in laying hens feeding was discussed. The extract from Spirulina sp. microalgae was produced by supercritical extraction with pure CO2, without the use of a co-solvent. The applicability of the preparations was tested on laying hens (120 hens, Lohman Brown, 36 weeks of life), housing in the deep litter system. Birds were divided into 4 groups (control, microalgae, microalgae extract or post extraction residue), where marine algae formulations were added to the feed or drinking water. The effect of the preparations on performance, egg traits (egg mass, eggshell strength and thickness, yolk color, albumen high) and fatty acids composition of egg yolk was examined. Also, a consumer questionnaire was undertaken to investigate the sensory characteristics of the eggs. The results demonstrated that extract from microalgae had a beneficial effect on eggshell strength, egg weight and yolk color. Consumers indicated that in the group, where algae extract was applied, the smell, general appearance, texture of yolk and albumen were improved but microalgae extract negatively affects the taste of eggs. Moreover, the dietary supplementation with microalgae extract did not significantly affect the n-3 fatty acids content of the eggs. There is a need for further research studies to examine the use of higher concentration of algae extract.

In: Innovative Bio-Products for Agriculture ISBN: 978-1-63485-558-7 Editors: K. Chojnacka and I. Michalak © 2016 Nova Science Publishers, Inc.

Chapter 1

INNOVATIVE TECHNOLOGY OF ALGAL EXTRACTS OBTAINED BY SUPERCRITICAL FLUID EXTRACTION USEFUL IN THE PRODUCTS FOR PLANTS, ANIMALS AND HUMAN Izabela Michalak* and Katarzyna Chojnacka Department of Advanced Material Technologies, Faculty of Chemistry, Wrocław University of Science and Technology, Smoluchowskiego, Wrocław, Poland

ABSTRACT In the present chapter, supercritical fluid extraction of seaweed collected from the Baltic Sea (Polysiphonia, Ulva and Cladophora) and microalga Spirulina (Arthrospira) was described. This novel extraction technique was discussed in terms of the isolation from the algal biomass of antioxidants (e.g., polyphenols), lipids (including polyunsaturated fatty acids), elements (micro-, macro- and toxic elelemnts), as well as plant growth hormones. On the basis of the composition of the obtained *

Corresponding author: Izabela Michalak. Department of Advanced Material Technologies, Faculty of Chemistry, Wrocław University of Science and Technology, Smoluchowskiego 25, 50-372 Wrocław, Poland. Phone: +48 713202434, fax: +48 713203469, E-mail: [email protected].

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Izabela Michalak and Katarzyna Chojnacka extract, their potential applications were proposed. Supercritical algal extracts can constitute a component of the products for plants (biostimulants of plant growth), animals (feed additives) and for human (cosmetics, diet supplement, pharmaceuticals, nutraceuticals). This wide application is possible due to the character of the extract – it is free of the organic solvents, which are commonly used in the clasical solvent extrcation.

Keywords: algae, extraction, polyphenols, plant growth polyunsaturated fatty acids, micro- and macroelements

hormones,

INTRODUCTION Algae are known as a rich source of biologically active compounds, such as: carbohydrates, proteins, minerals, lipids, polyunsaturated fatty acids, as well as bioactive compounds, such as antioxidants (polyphenols, vitamin E, vitamin C, mycosporine-like amino acids) and pigments, such as carotenoids (carotene xanthophyll), chlorophylls, and phycobilins (phycocyanin, phycoerythrin) [1]. Different extraction techniques can be used for their isolation, for example, traditional methods (extraction in Soxhlet apparatus, solid–liquid extraction and liquid–liquid extraction) and novel extraction techniques such as supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), enzyme-assisted extraction (EAE), pressurized liquid extraction (PLE) [2]. The increasing interest in the algal biomass, as a raw material for the extraction of active compounds, has been observed since 2000. In the last six years (2010-2016), a special attention was paid to the biomass of microalgae. According to the Web of Knowledge (February 09, 2016; http://apps.webofknowledge.com/), the number of scientific papers which have in the topic words “extraction of microalgae” is higher by 75% than papers with words “extraction of seaweed,” however such a big difference is observed in the last six years (Figure 1). As it can be seen from Figure 1, the number of papers that describe extraction of algae increased from decade to decade. In Table 1, the number of scientific papers concerning the extraction techniques of microalgae and seaweeds is summarised. Generally, the papers on the extraction of algae were written mainly in the last six years. Among the new techniques, the most often described is supercritical fluid extraction (SFE). The main attention is paid to the biomass of microalgae. It can be seen that other novel extraction techniques, such as: microwave-, enzyme-,

Innovative Technology of Algal Extracts …

3

ultrasound-assisted extraction (MAE, EAE, UAE, respectively), as well as pressurized liquid extraction (PLE) of microalgae and seaweed has been described in the literature mainly in the last six years. In the present chapter, a main attention was paid to the extraction of polyphenols, unsaturated fatty acids, plant growth hormones and microand macroelements from the biomass of commercially available microalga Spirulina sp. and from the Baltic green seaweeds, since they constitue a problem on the Polish coast, especially in tourist resorts. The method of utilization of this biomass was proposed. Supercritical fluid extraction was used for the production of algal extracts that can be used as plant growth biostimulants, as a component of feed additives or as an ingredient of cosmetics.

Number of scientific papers (according to Web of Knowledge)

1400 1285 1200

1000

extraction of microalgae extraction of seaweed

800

734

600 447 400 281 174

200 70

62 10

0 0

1

2

3

39

1 4

5

6

Years 1: 2010-2016; 2: 2000-2010; 3: 1990-2000; 4: 1980-1990; 5: 1970-1980

According to the Web of Knowledge; February 02, 2016. Figure 1. The number of scientific papers focusing on the extraction of algae in the last 36 years.

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Table 1. The number of scientific papers on the extraction techniques of microalgae and seaweeds Words in the topic of a scientific Number of papers paper in years 1926-2016 extraction and seaweed 1 412 extraction and microalgae 1 577 solvent extraction and seaweed 232 solvent extraction and microalgae 485 supercritical fluid extraction and 39 seaweed supercritical fluid extraction and 124 microalgae microwave assisted extraction and 58 seaweed microwave assisted extraction and 44 microalgae enzyme assisted extraction and 25 seaweed enzyme assisted extraction and 8 microalgae ultrasound assisted extraction and 19 seaweed ultrasound assisted extraction and 37 microalgae pressurized liquid extraction and 22 seaweed pressurized liquid extraction and 29 microalgae According to the Web of Knowledge; February 02, 2016.

Number of papers in years 2000-2016 1 146 1 510 205 472 38 120 58 44 25 8 19 37 22 29

SELECTION OF RAW MATERIAL FOR THE PRODUCTION OF ALGAL EXTRACTS Microalgae The demand for algal biomass is constantly growing. Algae are collected not only from the aqueous reservoirs (salt and freshwater), but they are also

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cultivated in artificial systems. This solution is especially designed for microalgae because it guarantees a constant chemical composition. The cultivation of microalgae can be carried out in several ways, depending on the algal species, environmental constrains, production volumes required and desired end-products (food, feed additive, energy source) [3]. Commercial cultivation of microalgae takes place in open ponds, raceway ponds or in closed photobioreactors [3-5]. The solar irradiance and temperature are key factors that influence the growth of microalgae. For the region of Baltc Sea, the environmental conditions are unfavourable for the commercial cultivation of microalgae [3]. In the recent years, the main interest in microalgae has resulted from their utilization for lipid extraction for biodiesel production. According to the Web of Knowledge (February 10, 2016), the number of scientific papers which have in the topic words “extraction of lipids” and “microalgae” increased significantly in the last 16 years: 1980-1990: 2 papers, 1990-2000: 19 papers, 2000-2010: 100 papers, 2010-2016: 777 papers. The main species of microalgae that are subjected to the lipid extraction are: Chlamydomonas reinhardtii, Botryococcus sp., Chlorella vulgaris and Scenedesmus sp., Shizochytrium limacinum, Pavlova sp., Nannochloropsis oculata, Synechocystis PCC 6803 [6, 7].

Macroalgae Macroalgae, which can be directly collected from the sea or cultivated in the seashore, have commercial market as functional food, feed, fertilizers for organic farming, pharmaceuticals, cosmeceuticals, phycocolloids, chemicals, biomass for energy, as source of biofuels [3, 8]. At the same time, large amounts of beach-cast seaweeds that result from the eutrophication are an environmental problem (e.g., emission of odours due to decomposition) in Baltic coastal areas, especially in tourist resorts [9]. For example, according to the report of the WAB Project (Wetlands Algae Biogas; http://wabproject.pl), 400 tons of algal biomass was collected only from the beach (164 tons) and sea (231 tons) in Sopot (Poland) in July, August and September 2011. These macroalgae are removed from the beach by the municipal services and disposed of on land allocated for that purpose [10]. By the collection of macroalgae from the beach or from the sea, not only the amount of nutrients causing eutrophication decreased [3], but also some usuful algae-based products can be obtained.

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The main species of marine macroalgae that were collected from the Baltic Sea in the Sopot city in 2013 involved: red alga (Rhodophyta): Polysiphonia and two green (Chlorophyta): Ulva and Cladophora [11]. Macroalgae species identified on the Sopot beach in previous years 2004-2006 belonged to three phylum – brown algae (Phaeophyta): Ceramium spp., Polysiphonia spp., Phyllophora brodiaei; red algae: Pilayella littoralis, Ectocarpus spp. and green algae: Cladophora spp., Enteromorpha spp., Ulotrix sp., Stigeoclonium spp. [10]. The use of macroalgal biomass from the Baltic Sea is very limited. However, there are some studies focusing on the utilization of macroalgae in this region, for example: for the energy production in Denmark [12], for the production of biological feed additives for animals [13, 14], or for biofertilizer [10].

DEVELOPMENT OF A METHOD OF RAW MATERIAL PREPARATION FOR THE PRODUCTION OF ALGAL EXTRACTS BY SUPERCRITICAL FLUID EXTRACTION Pretreatment of algal biomass before extraction process is a necessary step which improves extraction yield. For this reson, several pretreatment methods aimed at cell disruption were applied. Mechanical-physical pretreatment involves: autoclaving, bead-beating, microwaves, sonication, homogenization, freeze-drying, mechanical crushing, lyophilization, pulsed electric field technology, steam explosion, soaking plus steam explosion, steam flashing without soaking algal biomass, ultrasonication, conventional bubbling ozonation, pressure-assisted ozonation. In the chemical pretreatment, different chemicals are used, for example: HCl, NaOH, HNO3, H2SO4, CH3COOH, liquid nitrogen. Another option is enzymatic pretreatment with enzymes such as: cellulase, protease, driselase, alginate lyase S, viscozyme [2]. In the review of Crampon et al. (2011) it was shown that the main pretreatment method before supercritical CO2 extraction of oil from microalgae was freeze-drying and crushing to the desired size [15]. Mendes et al. (2003) proved that the form of microalga – Chlorella vulgaris (whole, crushed and slightly crushed) that is subjected to the supercritical fluid extraction influenced the extraction yield of lipids and carotenoids. It was found that the final yield of those compounds increased with the degree of crushing of the microalga cells. The

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crushing is important, because the cell wall is structurally a polymer what makes the extraction of the intracellular compounds difficult [16]. In the case of the algal biomass that is collected from the natural reservoirs (marine or freshwater), additional pretreatment step is required in order to clean the biomass. It involves washing the biomass several times with water (immediately after collection) to remove the adhering sand particles and impurities, drying and milling to obtain a homogeneous sample [2]. Drying at low temperatures (close to 35°C) is required in order to avoid the degradation of thermolabile compounds [15]. The discussed in the present chapter biomass of Baltic macroalgae, harvested directly form the sea (see Wilk et al., 2014 for details [17]), was subjected to the several steps before supercritical fluid extraction. After washing and drying, the biomass was prepared in three forms: coarse-grained grist obtained after preliminary grinding, fine-grained grist obtained after siving the ground biomass (coarse-grained grist) with a 0.5 mm sieve and pellets obtained after using a granulator. The fine-grained algal grist gave the best results in terms of the mass of the produced extract [11].

DEVELOPMENT OF THE CONCEPT OF MANUFACTURING TECHNOLOGY OF ALGAL EXTRACTS FOR A VARIETY OF APPLICATIONS Supercritical fluid extraction with CO2 of biologically active compounds from algae was discussed. This method has the advantages over other extraction techniques, because it has fast extraction rate, preserves heat labile components and during this process, solvent-free product is obtained [2, 18]. The main drawback of CO2 applied in SFE is its low polarity. This problem can be overcome by the addition of polar modifiers (co-solvents) that change the polarity of the supercritical fluid and increase its solvating power towards the analyte of interest. The addition of relatively small percentages (1-10%) of methanol/ethanol to carbon dioxide expands its extraction range to include more polar analytes [18]. The supercritical extraction of biologically active compounds from algae depends on many conditions – mainly pressure and temperature [15, 19, 20]. In the work of Qiuhui (1999), it was found that when the temperature is fixed, with the increase of pressure, the thickness of the supercritical CO2 increases and the capacity of CO2 dissolution increases. It was shown that the extraction

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of lipids from Spirulina platensis increased when the pressure increased from 300 to 350 bar. However, when the pressure increased from 350 to 400 bar, the rate of lipid extraction was reduced. For the fixed extraction pressure, the longer extraction time (1-4 h), the higher lipid rate [19]. Cheung et al. (1998) and Cheung (1999) showed that the conditions of supercritical fluid extraction with CO2 affected not only the fatty acid content, but also the composition of the algal lipid extracts. At lower pressure, more saturated fatty acids were extracted. When the pressure and density of fluid increased, the amount of unsaturated compounds and degree of unsaturation increased [21, 22]. The supercritical fluid extraction of algal biomass is mainly used for the isolation of lipids and pigments from the biomass of seaweeds and lipids, pigments (carotenoids: β-carotene; xanthophyll: astaxantin, fucoxanthin, zeaxanthin, β-cryptoxanthin, lutein; chlorophylls), and vitamins (e.g., E) from the biomass of microalgae [2, 15]. The supercritical fluid extraction of the biologically active compounds from the biomass of Baltic seaweeds and microalga Spirulina sp. was performed here in a closed-loop pilot plant comprising of two extractors each with a capacity of 40 L (see Rój (2014) for further details [23]). The experiments were carried out at 500 bar pressure and in the temperature of 40°C, as these conditions are suitable for the industrial processing of algae. The flow rate of CO2 was 200 kg/h [11]. In the previous paper it was found that the addition of co-solvent (ethanol) to CO2 during SFE (40°C, pressure 300-900 bar) of brown algaa (Fucus spp.) increased the extraction efficiency (up to more than 5.0%) [24]. The summary of upstream and downstream processing in the technology of supercritical algal extracts is presented in Figure 2 (based on [25, 26]).

DEVELOPMENT OF A METHOD OF THE POST-EXTRACTION RESIDUE MANAGEMENT – AS FERTILIZER OR BIOSORBENT During extraction process of algae, post-extraction residues are generated. This biomass should be utilized in order to lower the total production cost of the extarcation of biologically active compounds from algae. There are different ideas for their utilization. Bryant et al. (2012) proposed the use of post-extracted algae residue as a livestock feed [27].

Innovative Technology of Algal Extracts …

Figure 2. The summary of upstream and downstream processing in the technology of supercritical algal extracts.

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Gao et al. (2012) suggested the use of the algal biomass residue after oil extraction as a fermentation nutrient source instead of high-cost nutrient yeast. This approach will allow to lower the total production cost of biodiesel [28]. Efficient anaerobic digestion of lipid-extracted microalgae residues for methane energy production was proposed by Zhao et al. (2014). The new concept involved the application of biosorption process of trace element ions to produce value-added products for agriculture [29]. Innovative components of fertilizers and dietary feed supplements can be produced by biosorption, where micronutrients are bound with the biological material [30]. In the previous studies it was shown that the biomass of different species of macroalgae (e.g., Pithophora varia Wille, Enteromorpha prolifera, Vaucheria sp.) can be quickly and easily enriched with minerals, essential in animal nutrition. A wide range of biomaterials (especially waste biomass) can be used in the biosorption due to the presence of functional groups such as: carboxyl, phosphate, hydroxyl, amine, sulfhydryl that provide their biosorption properties [31]. In the work of Tuhy et al. (2014, 2015a), seaweed post-extraction residues after supercritical CO2 extraction, were enriched via biosorption with Zn(II) ions and then applied as a component of fertilizers in the germination tests on garden cress (Lepidium sativum). It was shown that the enriched residue was characterized by higher bioavailability of Zn(II) ions to plants when compared to traditional fertilizers: zinc sulfate and Zn-EDTA. Moreover, the application of this preparation led to the biofortification of plants with Zn. The mass of plants fertilized with enriched residue was higher than in the control group [32, 33]. Tuhy et al. (2015b) examined also the effect of post-extraction residues of Spirulina platensis (after supercritical CO2 extraction) enriched with Zn(II), Mn(II), Cu(II) ions via biosorption as micronutrient fertilizer in field trials on maize. It was found that the grain of maize was biofortified in the tested microelements which are essential for plants, animals and humans. Therefore, the biofortified grain of maize can be used as a component of animal feed or human diet. It can be useful in the prevention from microelement deficiencies [34].

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EXTRACTION OF POLYPHENOLS, LIPIDS, ELEMENTS AND PLANT HORMONES FROM ALGAE In the present chapter, a special attention was paid to the superitical fluid extraction of polyphenols, lipids, nutritive elements and plant growth hormones from the biomass of microalga – Spirulina sp. and from Baltic seaweeds (Polysiphonia, Ulva and Cladophora).

Antioxidants The antioxidant properties have many biologically active compounds extracted from algae, for example: glutathione, vitamins (C, E), carotenoids (α and β-carotene), polyphenols (phlorotannin, catechin, phenolic acid, flavonoids, tannins, lignans), mycosporine-like amino acids [1]. The antioxidant activity of the algal extracts can be determined by DPPH (2,2diphenyl-1-picrylhydrazyl) radical scavenging assay and β-carotene bleaching method [35, 36]. The quantity of antioxidants and the quality of the extracts can be assessed by Folin–Ciocalteu assay and NMR analysis [35]. As it was mentioned above, in the case of supercritical fluid extraction the addition of co-solvent to CO2 enabled the isolation of polar compounds. In the work of Mendiola et al. (2005) it was found that the best extraction conditions for the extraction of antioxidants (polyphenols and pigments: zeaxanthin, chorophyll a, β-carotene) from Spirulina platensis were as follows: CO2 with 10% of modifier (ethanol) as extraction solvent, 55°C (extraction temperature) and 220 bar (extraction pressure) [37]. Mendiola et al. (2007), in the next work on the supercritical fluid extraction of antioxidants from Spirulina platensis reported that the best antioxidant extract was obtained when CO2 with 10% ethanol as co-solvent under intermediate pressure and temperature (220-320 bar, 55°C) was used. For pure CO2, higher pressures and temperatures (320 bar, 75°C) were needed [36]. Mallikarjun et al. (2015) noted that the phenolic content in the supercritical extract obtained with CO2 from Spirulina platensis (40°C, 120 bar) was found to be 3.4 mg/g [38]. In the work of Wang et al. (2007), supercritical extract from Spirulina platensis, obtained under optimum conditions (48°C, 200 bar, 4 h), contained also flavonoids, β-carotene, vitamin A and vitamin E (α-tocopherol), which may contribute greatly to its high antioxidant activity [39]. Mendiola et al. (2008) optimized supercritical fluid extraction to obtain fraction highly enriched with vitamin E from microalga

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Spirulina platensis. The predicted value of vitamin E concentration at the optimum conditions (83°C, 362 bar), provided by the statistical program was equal to 29 mg/g extract. This implied on a tocopherol enrichment of more than 12 times than its initial concentration in the raw material [40]. This shows that during supercritical fluid extraction, concentrate of biologically active compounds was obtained. The supercritical fluid extraction of antioxidants from seaweeds that are found in Baltic Sea (species Polysiphonia, Ulva and Cladophora) has not been extensively studied, yet. Usually, for these algal species traditional solvent extraction is applied. Only in the work of Parjikolaei et al. (2014), SFE was used to isolate carotenoids from freeze-dried Ulva lactuca. The highest carotenoid content and total extract yield were obtained at 400 bars (examined range: 100 to 400 bars) and 55°C (examined range: 35 and 55°C). It was also found that SFE with CO2 was not as efficient as conventional extraction in pure ethanol. However, the addition of 5% of ethanol as co-solvent increased the total carotenoid content to 55 mg/kg of dried alga, which is 70% and 53% higher than the amount obtained using pure SFE-CO2 and ethanol, respectively [41]. Antioxidant activity of the water and ethanol extracts obtained from Ulva lactuca collected from Danish coast were examined in the work of Farvin and Jacobsen (2013). The total phenolic content in the water extract was 2.24 mg/g seaweed dry weight of GAE (gallic acid equivalents) and in the ethanol extract 2.37 mg/g. In the ethanol extract also α-tocopherol content was examined and for Ulva lactuca it was 2.7 µg/g of the extract [42]. In the literature, there are no papers concerning supercritical fluid extraction of antioxidants from Polysiphonia and Cladophora. In the case of Polysiphonia, antioxidant properties of the extract obtained only by solvent extraction in a Soxhlet extractor with the use of solvents methanol:chloroform (2:1) were examined. It was found that the total phenolic content in the crude extract was 72 mg/g dry weight of seaweed of GAE [43]. Horincar et al. (2011) examined total phenolics in extracts obtained with water, acetone, methanol, ethanol and hexane from Cladophora vagabunda. The best results were obtained for water extract and the total phenolics content was 2.1 mg/g seaweed dry weight of GAE [44]. In the frame of the present work, it was found that the supercritical extract of Baltic algae (mixture of Polysiphonia, Ulva and Cladophora) contained 20 mg/g of total phenolic compounds. The aqueous fraction of algal extraction contained 3.5 µg/ml of phenolic compounds [11].

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Lipids Lipid extraction methods involve mainly mechanical methods (oil expeller or press, ultrasound assisted and microwave assisted methods) and chemical methods (solvent extraction, supercritical CO2, ionic liquid extraction) [5-7]. In the recent years, a special attention has been paid to the supercritical fluid extraction of bioactive compounds from microalge. Among them, extraction of lipids (including polyunsaturated fatty acids) from Spirulina sp. is extensively studied. Mendes et al. (2003 and 2006) compared the supercritical CO2 extraction of γ-linolenic acid (GLA) and other lipids from Spirulina maxima with organic solvent extraction (ethanol, acetone, hexane and a mixture of chloroform, methanol and water). It was found that the addition of a polar co-solvent (ethanol) increased both lipid and GLA yields when compared to the extraction with pure CO2. Also the increase of pressure and temperature had a positive effect on the extraction of GLA. However, the supercritical extracts showed a low yield, when compared with those obtained with organic solvents. It is worth mentioning that Spirulina maxima can produce large amounts of GLA which also plays an an important role in the human metabolic pathway and therefore has a significant pharmaceutical importance [16, 45]. In the work of Wang et al. (2007) it was found that the main fatty acids in the extracts from Spirulina platensis isolated by SFE under optimum conditions (48°C, 200 bar, 4 h) were palmitic acid – 35%, α-linolenic acid (ALA, C18:3, n-3) – 22% and linoleic acid (LA, C18:2, n-6) – 21%. These components may contribute to the antioxidant activity of the obtained supercritical extract [39]. Qiuhui (1999) reported that the lipids extracted by SFE from Spirulina platensis could be used as additives to health foods containing γ-linolenic acid. The composition and the content of fatty acids in the extract obtained under optimal conditions (40°C, 400 bar, 4 h) was as follows: oleic acid (C18:1) – 12%, linoleic acid (LA, C18:2, n-6) – 36%, αlinolenic acid (ALA, C18:3, n-3) – 15%, γ-linolenic acid (GLA, C18:3, n-6) – 23% by weight of lipids [19]. Supercritical fluid extraction of lipids from green macroalgae was randomly studied. This could result from the low content of lipids in the raw biomass of seaweeds from Chlorophyta. For example, the content of lipids in Cladophora fracta was 14% (in dry mass), whereas in microalga – Chlorella protothecoides was 29% [46]. The lipid accumulation in the cells of microalgae can range from 25–80% of its dry weight – for example in microalga Schizochytrium spp., the oil content is 50–77% dry wt. Additionally,

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microalgae are characterized by high photosynthetic efficiency, high biomass production and fast growth, therefore are considered to be good candidates for biofuel production [6]. In the work of Messyasz et al. (2015), supercritical fluid extraction was used for the isolation of lipids from Cladophora. It was found that the fatty acid composition in Cladophora extract depended on the extraction method and solvent. The highest amounts of detected fatty acids, both saturated and unsaturated, were obtained using supercritical fluid extraction (45°C, 700 bar) rather than classic method of extraction (Soxhlet) with ethylene alcohol and acetone. The content of fatty acid (% weight) in the dry matter of the extract obtained with ethylene alcohol was 19%, with acetone 37% and with SFE 63%. The main fatty acids in the supercritical extracts were: stearidonic acid (SDA, 18:4, n-3) – 3.1% weight of fatty acids in dry matter of the extract, αlinolenic acid (ALA, 18:3, n-3) – 5.2%, linoleic acid (18:2, n-6) – 6.5%, oleic acid (18:1, n-9) – 9.3% [47].

Elements The extracts obtained by SFE are totally free of toxic metal ions since they are not extractable, even if they are present in the raw material. No heavy metals are present in CO2 or the equipment. This is one of the main advantages of the supercritical fluid extraction [48]. In the literature, the multielemental composition of algal extracts obtained by supercritical fluid extraction has not been studied so far. The first reports have appeared recently. Michalak et al. (2016 a) examined the content of the supercritical extract obtained from Baltic seaweed [11], whereas Świniarska et al. (2015) the extract from Spirulina sp. [49] (Table 2). It should be noted that toxic elements were extracted in small amounts from the raw algal biomass.

Plant Growth Hormones In many algae, phytohormones were detected in concentrations comparable with their content in higher plants. Moreover, the spectrum of their biological activities corresponds to the functions of higher plant hormones [50]. These compounds including auxin, gibberellins, cytokinins, ethylene, abscisic acid, polyamines and brassinosteroids are biochemical substances that control many physiological and biochemical processes in

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plants [51, 52]. In the literature it was noted that some of the listed plant hormones are present in green seaweeds, for example: auxins: indole-3-acetic acid – IAA (Enteromorpha, Cladophora, Caulerpa), gibberellins (Caulerpa), lunularic acid (Enteromorpha), polyamines (Ulva), as well as in microalgae: IAA (Chlorella), cytokinins (Protococcus, Chlorella, Scenedesmus, Chlamydomonas), abscisic acid – ABA (Chlorella, Dunaliella, Haematococcus), jasmonic acid – JA (Dunaliella, Chlorella), polyamines (Chlorella), brassinosteroids (Hydrodictyon) [50]. Table 2. Multielemental composition of the raw biomass and the algal extracts obtained by supercritical fluid extraction Element

Raw biomass of Extract from Raw biomass of Baltic seaweed Baltic seaweed Spirulina sp. [11] Own data (unpublished) mg/kg d.m. mg/L mg/kg d.m. Microelements B 190 ± 30