ALGAE DERIVED PRODUCTS AND TECHNOLOGIES

CHRISTOPHER J. CHUCK

University of Bath 1st October 2008

CONTACT DETAILS

UNIVERSITY OF BATH

Claverton Down, Bath, BA2 7AY

BIOLOGY

Professor Rod Scott [email protected] +44 (0)1225 383447

CHEMISTRY

Professor Matthew Davidson Dr. Christopher Chuck Head of Inorganic Chemistry Post Doctoral Research Officer [email protected] [email protected] +44 (0) 386443 +44 (0) 1225 384970

CHEMICAL ENGINEERING

Dr. Alexei Lapkin Senior Lecturer [email protected] +44 (0) 383369

MECHANICAL ENGINEERING

Professor J. Gary Hawley Dr. Chris Bannister Dean of Engineering RCUK Academic Fellow [email protected] [email protected] +44 (0) 1225 386855 +44 (0) 1225 384970

MANAGEMENT STRUCTURE

Dr. Miles Davis Research Portfolio Manager [email protected] +44 (0) 1225 384795

i UNIVERSITY OF THE WEST OF ENGLAND

Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY

SCHOOL OF LIFE SCIENCES

Dr. Stuart Shales Dr. Heather McDonald Senior Lecturer in Environmental Lecturer in Plant Science Biotechnology [email protected] [email protected] +44 (0) 117 32 82476

+44 (0) 117 3282490

MANAGEMENT STRUCTURE

Mr. David Lennard Deputy Head, Business and Community Division [email protected] +44 (0) 117 3282749

PLYMOUTH MARINE LABORATORY

Prospect Place, The Hoe, Plymouth, PL1 3DH

Mr. Stephen Skill Dr. Carole Llewellyn Biochemical Engineer Microbial Biogeochemist [email protected] [email protected] +44 (0)1752 633100 +44 (0)1752 633100

ii TABLE OF CONTENTS

CHAPTER 1: AN INTRODUCTION TO ALGACULTURE 1

1.1 Introduction 1 1.1.1 Algal Classes 4 1.2 Algaculture 13 1.2.1 History 13 1.2.2 Current Production of Algae 13 1.3 Growth Parameters 15 1.3.1 Medium and Nutirents 15 1.3.2 Gas Transfer 17 1.3.3 Mixing 19 1.3.4 Light Requirements 21 1.4 Strategies for Yield Improvement 24 1.5 References 26

CHAPTER 2: BIOREACTOR DESIGN 31

2.1 Open Reactor Systems 32 2.1.1 Shallow Ponds 32 2.1.2 Tanks 33 2.1.3 Circular / Raceway Ponds 33 2.1.4 Sloped Outdoor Reactors 34 2.2 Closed Reactor Design 35 2.2.1 Tubular Reactors 35 2.2.2 Flat Plate Reactors 40 2.2.3 Fermenter Type Reactors 41 2.3 Processing 44 2.3.1 Separation of Microalgae 44 2.3.2 Processing Extracted Matter 46 2.4 References 48

iii CHAPTER 3: ALGAE DERIVED PRODUCTS AND TECHNOLOGIES 51

3.1 Pharmaceutical Products 52 3.1.1 Anthelmintic 52 3.1.2 Antibacterial 52 3.1.3 Anticancer 54 3.1.4 Anticoagulant 56 3.1.5 Antifungal 57 3.1.6 Antiplatelet 57 3.1.7 Antiprotozoal 57 3.1.8 Antiviral 58 3.2 Fine Chemical Products 60 3.2.1 Vitamins 60 3.2.2 Pigments 64 3.2.3 Amino Acids 67 3.3 Lipids 69 3.3.1 General Fatty Acid Content 71 3.3.2 The Effect of Nutrients and Environmental Limitations 77 on Lipid Level and Fatty Acid Profile 3.3.3 Hydrocarbon Content 89 3.3.4 Wax Esters 92 3.3.5 Sterols 92 3.4 Polymers 94 3.4.1 Polyols and Carbohydrates 94 3.4.2 Biopolymers from Algae 95 3.5 General Human and Animal Nutrition 97 3.6 Fuels 99 3.6.1 Direct Use 99 3.6.2 Bio-Oil 100 3.6.3 Biodiesel 102 3.6.4 Hydrocarbon Formation 105 3.6.5 Bioalcohols 106 3.6.6 Hydrogen 108

iv 3.6.7 Biogas by Anaerobic Digestion 109 3.6.8 Gasification 110 3.6.9 Microbial Fuel Cell 110 3.7 Miscellaneous Uses of Algae 111 3.7.1 Biofouling 111 3.7.2 Wastewater Treatment 111

3.7.3 CO2 sequestration 112 3.8 References 113

CHAPTER 4: GLOSSARY AND DEFINITIONS 125

CHAPTER 5: DIRECTORY 128

APPENDIX I: CHEMICAL STRUCTURES 130

v Chapter 1: Introduction

CHAPTER 1

AN INTRODUCTION TO ALGACULTURE

1.1 INTRODUCTION

Algae are photosynthetic, nonvascular plants that contain chlorophyll a and have simple reproductive structures. Algae are incredibly diverse and exist in almost all environments including marine, freshwater, soil and even deserts. This diversity is demonstrated in the variability in size and structure, Brown kelp for example can grow up to 70m in length where some unicellular algae can be as little as 1 micron in diameter. Some algae resemble animals in that they have the ability to ingest particulate food, where some resemble higher plants in that they have organs that superficially resemble roots and stems etc.1 There is an estimated 26,000 species of algae, of which around 15 are exploited commercially, with a further 100 or so species having been investigated for commercial potential.2 Classifying algae is far from trivial and dozens of schemes and methods for the classification of algae exist (and will exist as further genetic information is uncovered). All living things are placed into kingdoms, plants are then subdivided into divisions (the animal kingdoms are separated into phyla). Further subgroups are then Class, Order, Family, Genus and finally Species. All algal divisions end in -phyta and the classes in –phyceae (Table 1.1). Due to this uncertainty discussion of the Orders and Families of algae will be kept to a minimum in this report.

1 Chapter 1: Introduction

Domain3 Kingdom Divisions Classes Common Name Eubacteria Bacteria Cyanophyta Cyanophyceae Blue-green algae

Eukaryota Plantae Chlorophyta* Chlorophyceae Green algae (Charophyceae) (Charophytes) Euglenophyceae Euglenoids Rhodophyta Rhodophyceae Red algae Chromophyta Phaeophyceae Brown algae Chrysophyceae Golden algae Bacillariophyceae Diatoms Xanthophyceae Yellow-green algae Haptophyceae Haptophytes (Prymnesiophyta) Eustigmatophyceae† Eustigmatophytes Dinophyceae Dinoflagellates Cryptophyta Cryptophyceae Cryptophytes Table 1.1 Divisions of algae and the common names in wide use

Traditionally cyanobacteria (blue-green algae) have been included among the other algae divisions. However cyanobacteria are prokaryotic (they have no membrane bound organelles or a cell nucleus among other distinctions). By more modern divisions algae are solely eukaryotes (conducting photosynthesis by membrane bound orgnanelles). On a biotechnological footing cyanobacteria are highly similar to the eukaryotic algae producing much the same bioactive molecules and commercially relevant compounds. Generally most modern publications will refer to their studies of ‘algae and the cyanobacteria’. For simplicity, however, cyanobacteria will be included in the umbrella term algae in this review.

* Relatively recently this division has been split into the Bryopsidophyceae, Pedinophyceae, Pleurastrophyceae, Prasinophyceae, Trebouxiophyceae and Ulyophyceae as well as the three classes listed. All of these classes are labelled green algae and are referred to in this review as such. † Sometimes split into two classes (Prasinophyceae) collectively known as pico-plankton

2 Chapter 1: Introduction

Other terms are commonly used in relation to algae such as planktonic, benthos or thermophile these terms are not defined by a genetic classification but rather by the ecological niche that these organisms live in. These groupings can include any life which exists primarily in the pelagic zone (open sea), benthic zone (seabed) or organisms which live in or around hydrothermal vents. In the algae a broad range of cell types exist. Some algae are unicellular where some far more complex living in colonies or as intertwining filaments. Almost all algal cells are microscopic. A simple diagram of both a prokaryotic and eukaryotic cell is given in Figs. 1.1 and 1.2.

Fig. 1.1 Diagrammatic representation of a eukaryotic cell*

Fig. 1.2 Diagrammatic representation of a prokaryotic cell (Bacteria)*

* Image adapted from http://www.harweb.com/cell

3 Chapter 1: Introduction

1.1.1 ALGAL CLASSES

In this section a brief outline of each of the classes is given, all this information has been adapted from two sources unless otherwise stated.1, 4 As the variation within classes is huge, the information given below should be taken as a guide and does not necessarily apply to all algal species within that class.

1.1.1.1 Cyanophyceae – Blue-Green Algae (Cyanobacterium)

These algae are found in almost all environments including soil, marine and fresh water. The body type is generally simple and limited however examples of unicell, colonies, branched and unbranched filaments can be found. Almost all cyanobacteria have an outer layer of wall material called the sheath. This cell wall can soft or firm and can be composed of up to four layers some components found in the wall are simple sugars, muramic acid, glutamic acid, diaminopilmelic acid, galactosamine, glucosamine and alanine. Blue-green algae posses no flagellated cells (locomotive) and algae of this class move by gentle swaying or gliding motions. The algae have chlorophyll a, β-carotene and several xanthophylls yet it is the increased levels of phycobiliproteins (mainly phycocyanin) which gives this class its unique colouration. Nitrogen fixation (removal of atmospheric nitrogen) has been observed for some species of cyanobacteria. The main storage food product is a branched carbohydrate, glycogen.

Genera Species Notes Arthrospira Spirulina is the former name of the Genera, now no (Spirulina) platensis longer used scientifically yet still in use maxima colloquially, mainly used as a food source.5 Aphanizomenon flos-aquae β-carotene content, health food supplement.5 Table 1.2 Representative genera and species of the cyanobacteria

* Image taken from Era-Net; www.ict-science-to-society.org/Pathogenomics/Bacteria.htm

4 Chapter 1: Introduction

1.1.1.2 Chlorophyceae and Charophytes – Green Algae

Like the cyanobacteria, green algae are also found in almost all environments such as fresh water, the pelagic and bethnic marine zones, soils and even snow. Certain species are also known that can withstand extreme temperatures. Green algae are probably the most studied genera due to their wide distribution and appearance. Green algal unicells, colonies and filaments all exist as do membranous forms, multinucleate types and advanced algal types where the thallus is separated into nodes and internodes. The majority of unicells and colonies are microscopic, yet in some species can be as large as a meter long. Numerous types of filamentous algae are also found and a wide range of sizes is noted. Unlike the cyanobacteria, some unicells and colonies are motile. In most forms there is a cell wall generally comprised of cellulose, in the cases where cellulose is not found (algae such as Volvax sp.) pectin, sporopollenin, calcium carbonate or even silica has been observed. Reproduction of the green algae is extremely diverse across the class. Green algae consist of chlorophylls a and b, β-carotene as well as a range of xanthophylls. Unlike the cyanobacteria, the identity and level of these xanthophylls is highly dependant on the age, nutrients available and the species. The reserve food for this class of algae is generally true starch as well as triglycerides in some species.

5 Chapter 1: Introduction

Genera Species Notes Chlorella vulgaris Food supplement,6 fish food,7 triglycerides8-12 protothecoides pyrenoidosa Dunaliella salina Vitamin E,13, 14 β-carotene,15 food supplement,16 tertiolecta triglyceride content12, 17 bardawil primolecta Haematococcus pluvialis Astaxanthin production18 Muriellopsis Lutein19 Chlamydomonas reinhardtii Hydrogen20 Ankistrodesmus Triglyceride content Botryococcus braunii Lipid (hydrocarbon) content, filamentous10, 21 Chlorococcum oleofaciens Triglyceride content9 Neochloris oleobundans Triglyceride content12 Scenedesmus TR-84 Triglyceride content10 Stichococcus bacillaris Triglyceride content10, 12 Tetraselmis suecica Triglyceride content10, 22 Nannochloropsis oculata Marine plankton, high triglyceride content10, 23 Nannochloris triglyceride content10 Table 1.3 Representative genera and species of the green algae

1.1.1.3 Euglenophyceae – Euglenoids

Euglena exhibit both animal-like (a cell covering rather than wall) and plant-like (photosynthetic cells) characteristics, and are generally found in fresh water such as a river or stationary ponds, soil or brackish mud. They are commonly found when a large amount of organic material is present and will even bloom if this level is high enough. The Euglenoids generally require at least one vitamin to grow, usually vitamin B12. They occur mostly as flagellated unicells, and a large variation in both shapes and sizes is observed, even within a single organism. These unicells may sometimes be enclosed in a gelatinous matrix, forming a primitive colony yet this is an exception not the rule.

6 Chapter 1: Introduction

Euglenoids contain both chlorophyll a and b, β-carotene and several xanthophylls such as astaxanthin (enough to give some species a red tinge). The food reserves are generally paramylon, a linear carbohydrate. Euglenoids have no cell wall but rather a covering called a periplast or pellicle generally formed from protein strips which wrap spirally around the cell material, this differs to a cell ‘wall’ as the plasma membrane is still outside of this pellicle.

1.1.1.4 Rhodophyceae – Red Algae

Red algae can exist in numerous habitats but are generally found in the sea along coasts, especially in the tropics. Most are macroalgae with sizes ranging from 5 -25 cm but some species can grow up to 2m. A few well known red algae genera are uni- cellular (Prophyridium, Rhodella, Rhodosorus) and thus reproduce by cell division, mostly these cells will aggregate as the wall material adheres to other cells. Almost all other genera in this class are branched filamentous. The algae possess no flagellated or ciliated cells. Red algae contain chlorophyll a and d,* α- and β-carotene, several xanthophylls and the phycobiliproteins; R-phycocyanin and R-phycoerythrin. This latter component is generally the most prevalent and gives the algae a red tinge.† The reserve food is generally floridean starch and/or simple organic compounds.

Genera Species Notes Porphyridium Phycoerythrin production24, 25 Erythotrichia Bangia Table 1.5 Representative genera and species of the red algae

* Chlorophyll d only present in a limited number of advanced species † Not all red algae is red, in some species the dominant pigment is phycocyanin (blue/green)

7 Chapter 1: Introduction

1.1.1.5 Phaeophyceae – Brown Algae

No unicellular, simple filamentous or colonies of any type exist in this class. All the brown algae are large with sizes ranging from 1mm-70m long. There are many common names associated with the brown algae such as seaweed (including red algae) or kelp (Order Laminariales). The brown algae are the most complex of the algae and certain species resemble higher plants. Most brown algae produced adhesives or other elaborate holdfast systems are almost always found attached to rocks or other substrates. Brown algae are generally found in temperate marine habitats along the coast, though certain orders are also found in the tropics (e.g. the Sargasso sea) they are less prevalent there. Brown algae contain chlorophyll a and c, α- and β-carotene and several xanthophylls (flavoxanthin, violaxanthin and lutein). It is the first two xanthophylls which give the algae its colour, which is pretty standard across the whole class. The reserve foods are mannitol, laminarin and fats as well as alginic acid in the cell wall.

Genera Species Notes Ectocarpus Scytosiphon Laminaria Botrydium granulatium triglyceride content12 Table 1.6 Representative genera and species of the brown algae

1.1.1.6 Bacillariophyceae – Diatoms

Diatoms are found in fresh and marine water as well as soils. They are the major algal component of plankton and are frequently the bloom organisms, especially in winter. They also exist in and on, other algae, seaweed, seagrasses, animals and rocks. All diatoms are simple unicellular organisms which exist free living, in colonies or in filaments. Diatoms contain chlorophyll a and c, α- and β-carotene and several xanthophylls (diatoxanthin,* fucoxanthin and diadinoxanthin). The food reserves are

* Major component in most species

8 Chapter 1: Introduction mainly chrysolaminarin and leucosin though oil is also found in some Genera. The wall is composed of silica impregnated into pectin, there is generally no cellulose.

Genera Species Notes Chaetoceros Fish food, aqua culture26 Phaeodactylum Fish food, aqua culture,26 EPA content,27 triglyceride content10 Nitzschia palea EPA content27 triglyceride12 Biddulphia aurita Triglyceride content12 Cyclotella DI-35 triglyceride content10 Thalassiosira pseudonana triglyceride content28 Cylindrotheca fusiformis lipid (triglyceride and phospho-) content9 Hanzschia DI-160 triglyceride content10 Navicula pellisulsa triglyceride content12 Table 1.7 Representative genera and species of the diatoms

1.1.1.7 Xanthophyceae – Yellow-Green Algae

This class of algae is commonly found with the green algae in freshwater and marine habitats, they are typically unicellular and exist as unicells, colonies or filaments. This class is generally not motile. Yellow-green algae are vary rarely the dominant organism in any environment. The yellow-green algae contain chlorophyll a and sometimes c, β-carotene is found as are several xanthophylls (diadinoxanthin* and vaucheriaxanthin). The food reserves are largely unknown, yet triglycerides have been found in most of the genera examined as well as the carbohydrate chrysolaminarin. The wall tissue is mainly made up of cellulose and pectin

1.1.1.8 Chrysophyceae – Golden Algae

Chrysophytes are generally found in colder regions including northern marine habitats, mountain streams or in the winter. The golden algae grow well under ice in fresh water. This algal class is rarely the dominant class and a commonly found with

* Major component in most species

9 Chapter 1: Introduction other algal species, though some golden algal species can impart a ‘fishy’ odour and taste to water if their concentration becomes too high. In this class both autotropic, and hetetotropic growth is observed and varies widely across genera. Structurally there are few filamentous forms while unicellular, amoeboid and colonial forms are common, most organisms have a flagellated stage. All members have chlorophyll a (though a few also possess c), α- and β- carotene as well as a few xanthophylls including fucoxanthin. Storage products are a branched carbohydrate and lipid oil. Wall structure is highly variable across the class, some golden algae possess cellulose, elaborate silica skeletons or in some cases no wall at all.

Genera Species Notes Ochoromonas danniea triglyceride content12 Table 1.8 Representative genera and species of the golden algae

1.1.1.9 Haptophyceae

A highly debateable class of algae, is also sometimes termed a division. Haptophyta can include the class Prymnesiophyceae, or previously Haptophyta was counted as an order of the class Chrysophyceae. This class is so named as all species contain a haptonema (an external filiform appendage which bears a superficial resemblance to a flagellum). The Haptophytes are unicellular and form loose arrangements of cells which can be termed colonies or filaments depending on the conditions. The pigments found are generally the same as the golden algae. Little is known of the food reserve but it is probably a simple carbohydrate like chrysolaminarin or triglyceride rich lipids. Most haptophytes have external, unmineralized scales made of cellulose and protein, however, there are some genera which have walls comprised of calcium carbonate.

10 Chapter 1: Introduction

Genera Species Notes Pavlova lutheri Autotropic, used in mariculture hatcheries as food salina source7 Chrysochromulina triglyceride content12 Isochrysis galbana Used as food source for bivalve aqua culture.7 Table 1.9 Representative genera and species of the haptophytes

1.1.1.10 Eustigmatophyceae

This class of algae is also controversial as it is sometimes placed with the yellow- green algae. They mainly habit freshwater and sometimes soils though some species have been found in the plankton. All genera in this class are photosynthetic autotrophs and are unicellular in free form or as filamentous units. Eustigmatophyte only contain chlorophyll a, and β-carotene as well as several xanthophylls, with violaxanthin generally being the dominant compound. The food reserves are simple carbohydrates as discussed for the yellow-green algae as well as triglycerides in some species.

Genera Species Notes Nannochloropsis oculata High triglyceride content,10 EPA,27 gaditana Carotene content 29-31 Table 1.10 Representative genera and species of the Eustimatophytes

1.1.1.11 Dinophyceae – Dinoflagellates

Dinoflagellates (pronounced ‘dean’) are the organisms that have both animal-like and plant-like characteristics however all dinoflagellates have motile flagellate cells. There are a large amount of genera and species in this group and they exist in most habitats around the world but more so in the oceans. As organisms different species can be free living or symbiots found in marine life. The Dinophyceae are best known for their blooms called ‘red tide’ which can contain concentrations of millions of cells per ml as well as various toxins. The organisms contain chlorophyll a and c, β-carotene and several xanthophylls, especially peridinin and fucoxanthin, giving most of the algae a

11 Chapter 1: Introduction red/brown colouring. As a food reserve the algae store true starch and/or triglyceride lipids. Cell wall coverings are highly diverse across the class.

Genera Species Notes Peridinium cinctum triglyceride content12 Crypthecodinium cohnii DHA content32, 33 Table 1.11 Representative genera and species of the dinoflagellates

1.1.1.12 Cryptophyceae

The cryptophytes are a small collection of organisms consisting of around 24 genera. There are marine and freshwater types, which are generally motile when free living. The size varies but generally are small (up to 100 µm). Again this is a controversial classification and the Cryptophyceae are sometimes classed as an order in the class Dinophyceae. Or as a separate class with the Haptophyceae. The organisms themselves contain chlorophyll a and c but lack β-carotene instead they possess α-carotene and the xanthophyll alloxanthin. These algae can be green to red. The food reserves are starch and oils and most of the genera possess cellulose cell walls.

Genera Species Notes Rhodomonas salina Cryptomonas Table 1.12 Representative genera and species of the cryptophytes

12 Chapter 1: Introduction

1.2 ALGACULTURE

1.2.1 HISTORY

Microalgae have been used as a protein source intermittently by indigenous populations for centuries. This use as a protein source is limited to natural blooms and the industrial cultivation of microalgae only started after the second World War. Primarily algal biomass was examined as a source of protein, but soon this was expanded in to producing antibiotics and other biologically active chemicals.34

Interest grew with the development of algal technology to turn CO2 into O2 for space flights as well as in the use of waste water treatment.35 The fuel crisis in the 1970’s provoked a further interest in developing algae as an energy source, including the conversion of biomass into methane as well as a source of biodiesel.10 Commercial large scale operations to produce algae were started in the 1960’s with a Japanese firm producing Chlorella.36 This was followed by a large outdoor facility set in (a natural) Lake Texcoco in Mexico the culture grown here was Arthrospira.7 Within 20 years there were over 50 algaculture producing facilities worldwide, this did not just include Chlorella in Asia but also Dunaliella salina (mainly for β-carotene) in Whyalla Australia and other industrial plants in USA and Israel.5

1.2.2 CURRENT PRODUCTION OF ALGAE

The specific products produced by algae are extensively reviewed in Chapter 3. In this section a brief overview of the current market for algae is given. Currently the microalgal biomass market produces between 5000-7500 t of dry matter per year and generates approximately $1.25 x 109.37, 38 About nine tenths of algae cultivated worldwide is in Asia with most of the algae dry matter marketed as a nutritional supplement/additive.39 Mainly Spirulina, D. Salina and Aphanizomenon are used as a supplement being sold as tablets, capsules and as emulsions in liquids as a health food supplement or even for some clinical applications.40 Over a 1000 tonnes of Spirulina is produced annually with the USA and China being the largest producers.41 The largest single producer is Hainan Simai which grows 200 t dry matter a-1 in the Hainan province in China.

13 Chapter 1: Introduction

Other large facilities include Myanmar Spirulina Factory (Yangon Myanmar), Cyanotech (which produce 100 tonnes a-1) and Earthrise farms in California which also produces this strain and run a out door production facility which covers 440,000 m2.42

Fig. 1.3 The production facilities at Earthrise Farms (left) and the tubular reactor in Kötze, Germany (right)

Chlorella is produced in a large number of countries with over 70 companies world wide running production facilities. Chlorella is mainly used for the production of food additives and pharmaceuticals. The two largest production facilities are the Taiwan Chlorella Manufacturing Co. (Taipei) which produces around 400 tonnes of Chlorella a-1 and in Klötze, Germany where 150 tonnes a-1 are garnered from a tubular photobioreactor.43 D. Salina is produced primarily for β-carotene but can also be used in the cosmetic industry due to the production of glycerol (yet this is a side product and not economically feasible on its own). The largest facility is in Whyalla, Australia. Crypthecodinium cohnii is grown solely for its high DHA content by Martek in Columbuia, USA produces around 240 tonnes a-1 for baby food while Omegatech in the USA produces 10 tonnes.44

14 Chapter 1: Introduction

1.3 GROWTH PARAMETERS

For growth algae require water, a steady supply of macro- and micronutrients* as well as carbon (CO2 for all algae plus organic substrates for heterotrophic growth) and light. Algal growth is generally measured by the specific growth rate (h-1) which is the fraction increase in biomass over a unit time. When growing algae there will be a certain lag time initially where the specific growth rate will be sub maximum. This is due to the adjustment of the cells to the conditions and the presence of non-viable cells and other organisms. This is followed by an exponential (logarithmic) phase where cells grow and divide as an exponential function of time. This is then followed by a linear growth phase where the quantity of light energy absorbed is determined by the cell concentration and not the photon flux density.

1.3.1 MEDIUM AND NUTRIENTS

Algae are marine organisms and as such need to be grown in water. As discussed previously each algal strain has different elemental requirements, yet these elements are generally supplied as inorganic salts in the aqueous solution. The pH of solution is vital and is generally controlled by increasing the amount of CO2 in the solution (CO2 forms a weak acid in water and reacts with the alkaline elements). Elements can also be supplied as a gas (flue gas) and as such can be pumped into the system with the

CO2. Algae need a large amount of nutrients to be available for optimised growth. The identity and ratio of these differs depending on the algal strain (a summary of which is given below in Table 1.13). All algae need a certain amount of metal ions, anions and a nitrogen source. All of these elements are added as a solution in the water and complexing agents such as EDTA or citric acid are commonly used to ensure solubility.

* Macronutrients (10-3 – 10-1 M) include N, P, K, Mg, Na and Cl, micronutrients (10-7-10-3 M) include Fe, Cu, Zn, Mn, B and Mo

15 Chapter 1: Introduction

Chlorella Duniella Spirulina Porphyridium Scenedesmus

Growth mode AC HC AC HC AC HC AC HC AC HC Elements Na R - R - R - R R Mg R R R - R - R - R R K R R - R - R - R R Ca R - R - R - R R Fe R r R - R - R - R R Zn R r r - R - R* - R R Mn R - R - R* - R* R* Mo R† R r - - - R* R* Cu r r - - R* - R* R* B R R - - - Ni - - - R* R* Se - r - - Al - - - r r V r - - - R* R* Co r - - R* - R* R* 3- P (as PO4 ) R R R - R - R - R R 2- S (as SO4 ) R R R - R - R - R R Cl R R R - R - R - R R Nitrogen Source - - -

NH4 P P - - P - P P

NO3 P - P - P - Urea - - - P P Organic Carbon - - - Glycerol - - - S Acetate S - - - P Glucose S - P‡ - - S Table 1.13 Nutrient solutions necessary for growth of algae, sorted by genus, R = required, r = recommended but not necessary, S = strain specific, P = preferred, data adapted from several sources.4, 45

* Required but only in trace amounts † - Required only if NO3 is lone nitrogen source ‡ Though a phototroph only, glucose in the medium does enhance growth

16 Chapter 1: Introduction

Generally algae nutrient ‘packs’ or solutions are not made from combining the individual isolated nutrients but rather they are synthesised from water taken from a particular source and adapted according to necessity or plant matter is reduced down and solubilised to form the necessary nutrient solution.46, 47 For research purposes many different types of solutions are available and can either be purchased from specific companies or synthesised from readily available manuals in the laboratory.

1.3.2 GAS TRANSFER

1.3.2.1 CO2

Microalgal biomass is made up of nearly 50% carbon, and this element is the major nutrient used for cell growth. In aqueous solutions carbon may exist in several - 2- chemical forms; CO2 (aq) H2CO3, HCO3 , CO3 . The general consensus is that all algae strains show a preference to CO2 over the inorganic salts and the efficiency of converting the inorganic salts to sugars is low. The interconversion between all these 48 compounds is rapid and the uptake of CO2 is not a limiting factor in growth. Air itself only contains around 0.03% (v/v) of CO2 so any maximisation of area or extensive mixing will not raise the CO2 level in the algal broth. The usual method of 49 introduction is to bubble enriched CO2 air into the reactor.

When the CO2 is introduced into the reactor there will be a concentration gradient as the CO2 is consumed. It has been found that the mass transfer of CO2 from the gas phase to the cell phase is mainly controlled by resistance offered by the liquid film of the cell. The rate of CO2 uptake by the cells determines the rate of CO2 being injected into the reactor. The rate of mass transfer (NCO2) is approximately given by

NCO2 = kL a(CCO2L* - CCO2L)

where kL is the liquid-phase mass transfer coefficient, a is the specific area available for mass transfer, CCO2L* is the concentration of CO2 in the culture medium that would equilibriate its actual partial pressure on the gas side and CCO2L is the concentration of CO2 in the bulk of culture medium. The lumped together parameter kLa characterises the CO2 mass transfer capability of the reactor and determines whether a certain reactor will be able to sustain the growth of a particular strain. This

17 Chapter 1: Introduction parameter is reliant on the size of gas bubbles in the reactor, the flow rate and the materials used. There are two ways to introduce the gas into the reactor, bubbling and via a hollow fibre membrane. The choice of this technology also has a large effect on 49-53 CO2 uptake. It should also be noted that chemical interactions between the CO2 - and other compounds (H2O, OH , NH3) in the liquid phase can accelerate the absorption and that the CO2 uptake is not solely governed by the diffusion of the gas in to the liquid cell.53

The final point to consider is the method of delivery used to inject CO2 into the reactor. There are two methods designed to introduce the critical amount of CO2 the reaction medium: i) passive mode where a large culture/air interface ensures CO2 delivery or ii) an active mode where aeration technology is used to increase the contact area. Passive mode is used for massive open pond systems (50- 300 ha) and the permeation comes from the boundary between the air and the broth. Passive mode is also used via membranes where the gas can diffuse into the culture. These membranes are generally made out of microporous, gas permeable materials (like silicone) and are arranged in the reactor vessel as a coil or tube. This system of delivery is highly efficient, there are no losses of CO2 to the atmosphere and there is no reason to purify the CO2 before use as the gas is not in contact with the actual cellular matter.54 However, large amounts of tubing are required, high inner pressures are necessary in order to achieve the correct transfer rate, meaning thick membrane walls are necessary to avoid rupture and the nature of the membrane promotes bacterial growth in the culture which in turn risks contaminating the algal matter and reducing the contact area. These factors can be somewhat moderated by the use of microporous hollow-fibre membranes instead of silicone.50, 55 The most common method though is to use an active mode and either circulate the gas mixture in the broth (by injection at one point 56, 57 or bubbling the gas through at the bottom of the reactor58-60) or by using a gas exchanger.61 Bubbling gas enriched air into the reactor via a variety of methods is the most commonly used procedure. This can be done either at the bottom, through accumulation might be observed in the more stagnant regions this can be overcome by injecting at the sides or through the blades of the stirrer. All these methods suffer from losing a large proportion of the CO2 to the atmosphere as only a maximum of 10% of the gas can be uptaken. Gas exchange units are much more efficient and technically can saturate the

18 Chapter 1: Introduction

solution with CO2. Gas exchange units are more expensive and the ability to scale up has also been bought into question. 49, 62

A number of control systems for CO2 injection have been examined to control the amount of CO2 in the reactor and to maintain the correct pH. These systems tend to be unresponsive though, and model-based predictive software needs to be developed to allow an adequate amount of control.63

1.3.2.2 O2

Each mol of chlorophyll produces between 50-400 mol of oxygen per hour. The -1 expected per-cell oxygen production is therefore around 25-400 fmol O2 h . The basic equation for photosynthesis is given below.

Energy + CO2 + H2O ↔ Sugar + O2

If O2 build up occurs in the reactor then the equilibrium can be shifted to the left and a marked drop in biomass production is observed. For the majority of algae O2 concentrations above atmospheric limit photosynthesis while dissolved O2 49, 58 concentrations of 35 mg/ L can be toxic. Generally speaking even though O2 build up is a problem, light and CO2 requirements are the major limiting factor in the production of biomass.

O2 build up is not a problem in reactors which are open to the atmosphere and very few problems are described when producing algae in closed reactors on a small scale, presumably due to the effectiveness of the gas exchange mechanisms. Systems which are continuously stirred also show little build up, however, O2 build up is one of the largest problems faced on scaling up closed reactors especially in systems with a high area to volume ratio. Several methods have been described to limit this build up via degassing units however none of these units reach any where near 100% efficiency.64-68 Using aleovaler flat panels 59 or hollow-fibre membrane apparata50 also leads to lower oxygen build up.

1.3.3 MIXING

Mixing is also a limiting factor in the growth of the algae. Mixing is required to provide a uniform distribution of algae within the medium so that there are no light,

19 Chapter 1: Introduction nutrient or temperature gradients, it will also limit ‘mutual shading’ it has been claimed that continuous cell movement from light and dark zones is essential to guarantee high biomass yields. This is because in a dense culture light can only penetrate to around 2-5 cm and so vigorous mixing is required to ensure all the algae receives enough energy.62 Stirring algal broth can be a difficult task as not all cells respond in the same way and turbulence can often be random. This effect or inadequate mixing leads to ‘dead zones’ generally at the bottom of the reactor where the algal matter will start to deteriorate due to the prevailing anaerobic conditions. Inadequate mixing can also lead to clumping of cells into aggregates and hence, three-phase systems (gas-liquid- solid) which reduce the mass transfer. On the other hand if the mixing is maximised then the shear-induced injury of cells becomes likely.49, 69The turbulence can be measured by determining the Reynolds Number (Nr), a dimensionless number which relates the tube diameter, liquid velocity, liquid density and culture viscosity. Reynolds numbers of 3000 are recommended in order to give good mixing. There are three general systems used to circulate algal broth; Pumping, mechanical stirring and gas mixing. Most of the outdoor systems use distant pumps (centrifugal, rotary displacement, peristaltic or diaphragm) to propel the broth around the circuit. These systems cause high shear stresses proportional not just to the rotation speed but also hydrodynamic stresses related to the amount of circuits that the cells are forced to undergo. Baffles placed to create a more even turbulence can greatly reduce this shear stress.70 Gas mixing causes a lot less damage, especially in systems which use airlift units (air is forced into a vertical tube, decreasing the density of the algal broth and forcing it to rise, at the top the liquid naturally flows down a slope and a circuit is created).71 In these systems bubble formation occurs though and this can cause damage to the cells.72 In a closed fermenter type reactor the suspension can be agitated by mechanical stirring, the correct rotation speed is highly dependent on the algae used but to reduce the chance of further shear stress gas can be passed through the stirrer and into the mixture via perforated holes in the blades. All microalgae have different sensitivity to shear stress and it is highly dependent on the organism used as to the optimum operating conditions.4

20 Chapter 1: Introduction

1.3.4 LIGHT REQUIREMENTS

Light is the basic energy source and the largest limiting factor in algal biomass growth. The photosynthetic active region (PAR) is assumed to be around 45% in the wavelength range 400-700 nm.73 The photosynthetic efficiency (PE) is defined as the fraction of light that is eventually stored as chemical energy. There is no agreed standard method to measure this value. Most algal systems have PE in the region of 6% however algae has been grown which has achieved 40% of the PE. There are several equations which can be used to calculate the PE, firstly the illuminated surface area per unit volume of the culture (Aν) must be calculated, this will differ depending on the reactor. Pirt et al. gave the following equations for microbial-growth energy. µ q = + m YG

where q is the specific rate of light uptake, µ is the specific rate of growth, YG is the maximum growth yield on light and m is the maintenance coefficient.

φ ⋅ I ⋅ A q = 0 v X

where φ is the fraction of photosynthetically available light, I0 is the total incident radiation and X is the biomass concentration. Combination of these two equations (where m is practically 0) gives the equation below where µ . X (biomass output rate) can be calculated

µ ⋅ X = φ ⋅ I0 ⋅ Av ⋅YG

In a reactor operated under a constant Aν and a given I0 it is possible to adjust µ and X so as to achieve the maximum biomass yield (YG). The light available differs depending on the source, therefore for reactors which harness the suns energy only the maximum I0 can only be controlled by 74 orientating the reactor towards or away from the sun. In artificially lit reactors the I0

21 Chapter 1: Introduction can be more accurately controlled. Light energy available to each cell depends on a few factors and not just the intensity of the light. For example dense populations of algae will reduce light penetration, and light levels decrease exponentially with depth. Increasing the intensity of light increases the specific growth rate only up to a point, beyond which the growth rate will start to decrease. This phenomenon known as the photoinhibition and is the result of reversible damage to the photosynthetic apparatus.75 As detailed in Chapter 3, the biochemical profile of the algae will also dramatically change with a change in light intensity.76-78 Certain systems are available to monitor growth (based on flow injection analysis79 or light transmittance sensors80) which means the optimum amount of light for both the desired biochemical profile and growth rate can be achieved.

1.3.3.1 Artificial Lamps

As providing energy by artificial light is a large expense in the production of algae in closed systems, it is important that the both the wavelength range and intensity are suitable for production. Each algal strain will have different requirements depending on the desired products, however, such information has been calculated in reference to standard lamps. Two factors are necessary in determining these values. 1) The amount of photons emitted in the spectral region absorbed by the algae per unit electrical energy consumed 2) the fraction of photons intercepted by the algae that get converted into stored chemical energy. It is out of the scope of this review to go into the mathematics but a full account is given by Simmer et al.81

Fig, 1.4 Spectral range of direct sunlight, reflected sunlight (A) and a metal halide lamp (B) taken from Ogbonna et al.82

22 Chapter 1: Introduction

1.3.3.2 LED Light Source

To commercially exploit the products yielded by algae a cheap, durable, but above all else highly efficient light source needs to be used. Lamps have large spectral ranges (Fig 1.4) and a large amount of the frequency generated is not used (and even in the case of UV and IR light frequencies can reduce biomass productivity). Light emitting diodes (LED) are light, small have a very long life expectancy, and are highly efficient so (unlike lamps) do not produce large amounts of heat. LEDs have a half power band width of ~20-50 nm. A commonly given reason for the low amount of biomass produced in algaculture (1 g dry mass m-2 h-1 in outdoor ponds) has been the so called light- saturation effect. Further experimentation reveals that by using pulsing lights, namely LED displays where the photonic input rate can be matched more firmly to the rate limiting steps of photosynthesis then this can be increased. By increasing the photonic flux the bioproductivity will increase also, this relationship continues until the algae reaches its particular photosynthetic processing capability.83 This is termed the flux tolerance, above this point photons are dissipated as heat by non-photosynthetic processes. Empirical observation has deemed algae flux tolerance from between 200- 400 µmol photons m−2 s−1 when using natural light. This can be improved greatly and efficient accommodation of instantaneous (but discontinuous) flux values of 5,000 µmol photons m−2 s−1, without flux saturation, were demonstrated in pulsed LED experiments.84 Photobioreactors using LED technology have been reported of giving flux tolerance values of up to 8000 µmol photons m−2 s−1. It therefore appears that the rate limiting process for improving this tolerance and therefore productivity is to be able to match the time pattern, spectrum and intensity of a pulsed LED to the dark reaction kinetics (averages over the algal culture). This on the other hand means that if ultra dense cultures are necessary, rapid light and dark cycles (imposed hydrodynamically) need to be coupled with thin channels.85

23 Chapter 1: Introduction

1.4 STRATEGIES FOR YIELD IMPROVEMENT

The cost of producing algae can be significantly reduced by applying a biorefinery approach (where more than one component is isolated and sold), improving the capabilities of the microalgae through molecular level engineering or increase in yield via bioreactor design. A biorefinery uses every available component of the biomass raw material and as such the cost of production for any given product is lowered. In Chapter 3 a list of potential side products and uses for the algae is discussed. Reactor design is another area where significant gains in growth have been made recently. An extensive list of current technology is detailed in Chapter 2. The final method which could potentially be used to enhance productivity of the various aspects of algae growth is molecular level engineering.86 There are several aspects which need to be addressed though. These are:

• To increase photosynthetic efficiency, enabling increased biomass on light. • To enhance the growth rate of the biomass. • To enhance the level of target compound in the biomass • To improve the tolerance of the algae to more extreme conditions • To eliminate light saturation phenomenon, so that growth continues to respond to increasing light level • To reduce photoinhibition, so that growth rate is no longer reduced at midday light intensities that occur in different zones • To reduce the susceptibility to photoxidative cell damage

One method to help achieve these goals is to genetically engineer the algae. The absence of cell differentiation makes microalgae a much simpler system for manipulation than the higher plants. Little work has been done on the genetic manipulation of algae and methods successfully used for other systems have failed when applied to algae mainly because of the considerable evolutionary distance between algae and other organisms.38 In addition, allelic genes are usually absent because of the haploid nature of most vegetative stages of microalgae. Transformation systems had to be developed almost from the start, including techniques to introduce

24 Chapter 1: Introduction

DNA into algal cells with suitable promoters, new selectable maker genes and expression vectors. These have been fulfilled for Bacillariophyceae Phaedactylum, Chlorophyceae Chlamydomonas, Cyanophyceae Synechococcus and C. Synechosystis.87, 88 In addition, algae often have an unusual codon usage which requires even further adjustment before a successful transformation is possible.37 However, successful breakthroughs in transforming algae have been noted. Zaslavskaia et al. reported a heterologous expression of a functional glucose transporter in the obligate photoautotrophic diatom Phaeodactylum, which enabled the alga to grow on glucose in the dark.89 Another success in this area was the expression of mosquito larvicidal properties in certain species of cyanobacteria.90 Raja et al. listed the following factors to consider in the genetic manipulation of algae.38

1. The accumulation of valuable substances in algae via genetic transformation can only increase up to the point where cellular metabolism starts to be negatively affected 2. Transgenic algae potentially pose a considerable threat to the ecosystem and will most likely to be banned from the outdoor cultivations or otherwise be under strict regulation 3. Usually the transgenic cells exhibit less fitness than wild type and therefore cells that lose the newly introduced gene quickly outgrow the transformants. To prevent this, a constant selection pressure is necessary, by the addition of antibiotics, a potential public health hazard. Therefore, the prime field of genetic engineering will be an improved production of valuable products and bioactive compounds in closed culture systems.

It seems then that the prime focus for genetic engineering will be the improved production of valuable products in closed cultures, and that this manipulation should complement rather than substitute the screening process.

25 Chapter 1: Introduction

1.5 REFERENCES

1. F. R. Trainer, Introducing Phycology, Wiley, New York, 1978. 2. T. A. Norton, M. Melkonian and R. A. Andersen, Phycologia, 1996, 35, 308- 326. 3. C. R. Woese, O. Kandler and M. L. Wheelis, Proceedings of the National Academy of Sciences of the United States of America, 1990, 87, 4576-4579. 4. M. A. Borowitzka and L. J. Borowitzka, Micro-algal Biotechnology, University Press, Cambridge, 1988. 5. P. Spolaore, C. Joannis-Cassan, E. Duran and A. Isambert, Journal of Bioscience and Bioengineering, 2006, 101, 87-96. 6. E. W. Becker, Biotechnology Advances, 2007, 25, 207-210. 7. M. A. Borowitzka, Journal of Biotechnology, 1999, 70, 313-321. 8. W. Xiong, X. F. Li, J. Y. Xiang and Q. Y. Wu, Applied Microbiology and Biotechnology, 2008, 78, 29-36. 9. Z. Dubinsky, T. Berner and S. Aaronson, Biotechnology and Bioengineering Symposium, 1978, 8, 51-68. 10. J. Sheehan, A Look Back at the U.S. Department of Energy's Aquatic Species Program - Biodiesel from Algae, NREL, Colorado, 1998. 11. H. Xu, X. L. Miao and Q. Y. Wu, Journal of Biotechnology, 2006, 126, 499- 507. 12. N. Kosaric and J. Velikonja, Fems Microbiology Reviews, 1995, 16, 111-142. 13. E. C. Carballo-Cardenas, P. M. Tuan, M. Janssen and R. H. Wijffels, Biomolecular Engineering, 2003, 20, 139-147. 14. A. Salguero, B. de la Morena, J. Vigara, J. M. Vega, C. Vilchez and R. Leon, Biomolecular Engineering, 2003, 20, 249-253. 15. R. Leon, M. Martin, J. Vigara, C. Vilchez and J. M. Vega, Biomolecular Engineering, 2003, 20, 177-182. 16. M. A. Hejazi and R. H. Wijffels, Trends in Biotechnology, 2004, 22, 189-194. 17. K. Tsukahara and S. Sawayama, Journal of the Japan Petroleum Institute, 2005, 48, 251-259. 18. R. T. Lorenz and G. R. Cysewski, Trends in Biotechnology, 2000, 18, 160-167.

26 Chapter 1: Introduction

19. J. A. Del Campo, J. Moreno, H. Rodriguez, M. A. Vargas, J. Rivas and M. G. Guerrero, Journal of Biotechnology, 2000, 76, 51-59. 20. J. P. Kim, C. D. Kang, T. H. Park, M. S. Kim and S. J. Sim, International Journal of Hydrogen Energy, 2006, 31, 1585-1590. 21. A. Banerjee, R. Sharma, Y. Chisti and U. C. Banerjee, Critical Reviews in Biotechnology, 2002, 22, 245-279. 22. G. C. Zittelli, L. Rodolfi, N. Biondi and M. R. Tredici, Aquaculture, 2006, 261, 932-943. 23. Q. Hu, C. Zhang and M. Sommerfeld, Journal of Phycology, 2006, 42, 12-12. 24. J. Fabregas, D. Garcia, E. Morales, A. Dominguez and A. Otero, Journal of Fermentation and Bioengineering, 1998, 86, 477-481. 25. J. Fabregas, E. Morales, J. Aran and A. Otero, Bioresource Technology, 1998, 66, 19-24. 26. J. A. L. Elias, D. Voltolina, C. O. C. Ortega, B. B. R. Rodriguez, L. M. S. Gaxiola, B. C. Esquivel and M. Nieves, Aquacultural Engineering, 2003, 29, 155-164. 27. E. H. Belarbi, E. Molina and Y. Chisti, Process Biochemistry, 2000, 35, 951- 969. 28. M. R. Brown, G. A. Dunstan, S. J. Norwood and K. A. Miller, Journal of Phycology, 1996, 32, 64-73. 29. E. Forjan, M. A. Quintanilla, R. Leon, J. M. Vega and C. Vilchez, Biomolecular Engineering, 2003, 20, 64. 30. J. M. S. Rocha, J. E. C. Garcia and M. H. F. Henriques, Biomolecular Engineering, 2003, 20, 237-242. 31. L. Rodolfi, G. C. Zittelli, L. Barsanti, G. Rosati and M. R. Tredici, Biomolecular Engineering, 2003, 20, 243-248. 32. Y. Jiang and F. Chen, Journal of Industrial Microbiology & Biotechnology, 1999, 23, 508-513. 33. Y. Jiang, F. Chen and S. Z. Liang, Process Biochemistry, 1999, 34, 633-637. 34. M. A. Borowitzka, Journal of Applied Phycology, 1995, 7, 3-15. 35. J. Delanoue, G. Laliberte and D. Proulx, Journal of Applied Phycology, 1992, 4, 247-254. 36. H. Iwamoto, in Handbook of Microalgal Culture, ed. A. Richmond, Oxford, Editon edn., 2004, pp. 255-263.

27 Chapter 1: Introduction

37. O. Pulz and W. Gross, Applied Microbiology and Biotechnology, 2004, 65, 635-648. 38. R. Raja, S. Hemaiswarya, N. A. Kumar, S. Sridhar and R. Rengasamy, Critical Reviews in Microbiology, 2008, 34, 77-88. 39. Y. K. Lee, Journal of Applied Phycology, 1997, 9, 403-411. 40. K. E. Apt and P. W. Behrens, Journal of Phycology, 1999, 35, 215-226. 41. O. Ciferri and O. Tiboni, Annu. Rev. Microbiol., 1985, 39, 503-526. 42. www.earthrise.com, June 2008. 43. K. Steinberg, http://www.bioprodukte-steinberg.de/index.php?op=algenfarm, June 2008. 44. O. P. Ward and A. Singh, Process Biochemistry, 2005, 40, 3627-3652. 45. T. Oh-Hama and S. Miyachi, in Micro-Algal Biotechnology ed. M. A. Borowitzka, Cambridge University Press, Cambridge, Editon edn., 1988, pp. 3-27. 46. M. A. Borowitzka, Journal of Applied Phycology, 1992, 4, 267-279. 47. C. A. Shaw, ed. C. Chuck, Advetec Adavanced Environmental Technologies, Bath, Editon edn., 2008, p. On Nutrients for Microalgae. 48. J. C. Goldman, M. R. Dennett and C. B. Riley, Biotechnology and Bioengineering, 1981, 23, 995-1014. 49. A. P. Carvalho, L. A. Meireles and F. X. Malcata, Biotechnology Progress, 2006, 22, 1490-1506. 50. B. S. Ferreira, H. L. Fernandes, A. Reis and M. Mateus, Journal of Chemical Technology and Biotechnology, 1998, 71, 61-70. 51. J. C. Goldman, M. R. Dennett and C. B. Riley, Biotechnology and Bioengineering, 1981, 23, 995-1014. 52. E. M. Grima, J. A. S. Perez, F. G. Camacho and A. R. Medina, Journal of Chemical Technology and Biotechnology, 1993, 56, 329-337. 53. P. Talbot, M. P. Gortares, R. W. Lencki and J. Delanoue, Biotechnology and Bioengineering, 1991, 37, 834-842. 54. Y. K. Lee and H. K. Hing, Applied Microbiology and Biotechnology, 1989, 31, 298-301. 55. A. P. Carvalho and F. X. Malcata, Biotechnology Progress, 2001, 17, 265-272. 56. G. C. Zittelli, F. Lavista, A. Bastianini, L. Rodolfi, M. Vincenzini and M. R. Tredici, Journal of Biotechnology, 1999, 70, 299-312.

28 Chapter 1: Introduction

57. E. M. Grima, J. A. S. Perez, F. G. Camacho, J. L. G. Sanchez, F. G. A. Fernandez and D. L. Alonso, Journal of Biotechnology, 1994, 37, 159-166. 58. A. S. Miron, A. C. Gomez, F. G. Camacho, E. M. Grima and Y. Chisti, Journal of Biotechnology, 1999, 70, 249-270. 59. M. R. Tredici, P. Carlozzi, G. C. Zittelli and R. Materassi, Bioresource Technology, 1991, 38, 153-159. 60. M. C. Garcia-Malea Lopez, E. Del Rio Sanchez, J. L. Casas Lopez, F. G. Acien Fernandez, J. M. Fernandez Sevilla, J. Rivas, M. G. Guerrero and E. Molina Grima, Journal of Biotechnology, 2006, 123, 329-342. 61. T. Stadler, Algal Biotechnology, Elsevier, New York, 1988. 62. E. W. Becker, Microalgae: Biotechnology and Microbiology, Cambridge University Press, New York, 1994. 63. J. L. G. Sanchez, M. Berenguel, F. Rodriguez, J. M. F. Sevilla, C. B. Alias and F. G. A. Fernandez, Biotechnology and Bioengineering, 2003, 84, 533-543. 64. M. Morita, Y. Watanabe and H. Saiki, Biotechnology and Bioengineering, 2000, 69, 693-698. 65. S. J. Pirt, Y. K. Lee, M. R. Walach, M. W. Pirt, H. H. M. Balyuzi and M. J. Bazin, Journal of Chemical Technology and Biotechnology B-Biotechnology, 1983, 33, 35-58. 66. S. J. Pirt, Journal of Chemical Technology and Biotechnology, 1982, 32, 198- 202. 67. M. A. Borowitzka, Journal of Marine Biotechnology, 1996, 4, 185-191. 68. E. M. Grima, F. G. A. Fernandez, F. G. Camacho and Y. Chisti, Journal of Biotechnology, 1999, 70, 231-247. 69. A. K. Panda, S. Mishra, V. S. Bisaria and S. S. Bhojwani, Enzyme and Microbial Technology, 1989, 11, 386-397. 70. C. Gudin and D. Chaumont, Bioresource Technology, 1991, 38, 145-151. 71. X. Zhang, B. C. Zhou, Y. P. Zhang, Z. L. Cai, W. Cong and O. Y. Fan, Biotechnology Letters, 2002, 24, 1767-1771. 72. M. J. Barbosa, Hadiyanto and R. H. Wijffels, Biotechnology and Bioengineering, 2004, 85, 78-85. 73. S. J. Pirt, Y. K. Lee, A. Richmond and M. W. Pirt, Journal of Chemical Technology and Biotechnology, 1980, 30, 25-34.

29 Chapter 1: Introduction

74. Y. K. Lee, S. Y. Ding, C. S. Low, Y. C. Chang, W. L. Forday and P. C. Chew, Journal of Applied Phycology, 1995, 7, 47-51. 75. F. Camacho Rubio, F. Garcia Camacho, J. M. Fernandez Sevilla, Y. Chisti and E. Molina Grima, Biotechnology and Bioengineering, 2003, 81, 459-473. 76. E. M. Grima, J. A. S. Perez, F. G. Camacho, J. L. G. Sanchez and D. L. Alonso, Applied Microbiology and Biotechnology, 1993, 38, 599-605. 77. E. M. Grima, F. G. Camacho, J. A. S. Perez and J. L. G. Sanchez, Process Biochemistry, 1994, 29, 119-126. 78. I. Perner-Nochta and C. Posten, Journal of Biotechnology, 2007, 131, 276-285. 79. L. A. Meireles, J. L. Azevedo, J. P. Cunha and F. X. Malcata, Biotechnology Progress, 2002, 18, 1387-1391. 80. J. M. Sandnes, T. Ringstad, D. Wenner, P. H. Heyerdahl, I. Kallqvist and H. R. Gislerod, Journal of Biotechnology, 2006, 122, 209-215. 81. J. Simmer, V. Tichy and J. Doucha, Journal of Applied Phycology, 1994, 6, 309-313. 82. J. C. Ogbonna, T. Soejima and H. Tanaka, Journal of Biotechnology, 1999, 70, 289-297. 83. H. P. Luo and M. H. Al-Dahhan, Biotechnology and Bioengineering, 2004, 85, 382-393. 84. D. J. Tennessen, R. J. Bula and T. D. Sharkey, Photosynthesis Research, 1995, 44, 261-269. 85. J. M. Gordon and J. E. W. Polle, Applied Microbiology and Biotechnology, 2007, 76, 969-975. 86. Y. Chisti, Biotechnology Advances, 2007, 25, 294-306. 87. K. L. Kindle, K. L. Richards and D. B. Stern, Proceedings of the National Academy of Sciences of the United States of America, 1991, 88, 1721-1725. 88. K. E. Apt and P. W. Behrens, Journal of Phycology, 1999, 35, 215-226. 89. L. A. Zaslavskaia, J. C. Lippmeier, C. Shih, D. Ehrhardt, A. R. Grossman and K. E. Apt, Science, 2001, 292, 2073-2075. 90. S. Boussiba, X. Q. Wu, E. Ben-Dov, A. Zarka and A. Zaritsky, Journal of Applied Phycology, 2000, 12, 461-467.

30 Chapter 2: Bioreactor Design

CHAPTER 2

BIOREACTOR DESIGN

For the intensive production of algae, two basic design categories exist; open systems, where most if not all the algal matter is exposed to the atmosphere and closed systems where the culture has no direct contact with the atmosphere.91 There are many considerations to be taken into account on determining the correct system to use. These factors include the strain of algae, the availability of land, water and nutrients, the climate, the cost of energy and the harvesting method.92 Other factors that also need to be considered are the light utilisation efficiency, ability to control environmental factors, the hydrodynamic stress placed on the algae and ability to maintain an axenic population as well as the potential for scale up.93 The final choice of reactor type is generally a compromise between optimising these considerations to achieve a viable outcome.

31 Chapter 2: Bioreactor Design

2.1 OPEN REACTOR SYSTEMS

Open systems were the first systems proposed for the production of algae and are still the most widely applied system for the production of algae presently. The reason for this is mainly economic, closed culture systems are very expensive and have proven difficult to scale up, this is coupled with the high energy costs of the artificial lighting necessary for indoor growth.94 Specific types of out door system are detailed below, however, open systems all have a number of constraints which limit the productivity. Firstly it is almost impossible to limit contamination of the selected strains with economically useless strains of algae and other microorganisms which feed off the algal matter. In order to avoid this, strains which require highly selective conditions such as a high pH or high salinity can be used. This guarantees the dominance of the required strain yet severely restricts the choice of algal matter. It is also impossible to keep the culture environment constant, and environmental factors play a large part in determining the processing parameters. The final general problem with using open systems is the cost of harvesting. The density of the algal matter tends to be far lower in an open system than a closed one, this means that the volume of material that has to be processed is enormous and substantially increases the production and therefore final cost of the products derived from the algal matter.95

2.1.1 SHALLOW PONDS

Algae can be produced in large open ponds which can either be naturally occurring or man made. To make an economical production process the ponds must have easy access to sea water and other nutrients, all year round sunshine and low land costs. For these reasons open shallow pond systems are not truly applicable in countries such as Israel, the UK or the USA, where land is too expensive and the climatic conditions too variable. Pool areas tend to be large for example in Whyalla, Australia Betatene Ltd. have been growing Dunaliella salina in ponds of up to 300 ha in area, the algal matter growing in the ponds was unmixed except by the wind and convection currents.96 Environmental conditions can be highly variable and this coupled with the depth of the pond, means that the growth of algae in shallow big ponds never reaches the theoretical maximum. Generally for large open ponds a depth of around 50 cm is

32 Chapter 2: Bioreactor Design ideal, this is deep enough to allow some confection mixing, and deep enough so that evaporation does not cause a salt imbalance yet shallow enough to allow adequate light for the cells. Delivering nutrients to the algae is also problematic in large open ponds, and the addition of CO2 can be highly inefficient. The maximum density of biomass achievable in open ponds is therefore very low with values normally falling between 0.1-0.5 g L-1. As this density is very low, the volumes needed to be processed are enormous for example at the Whyalla production facility up to a million litres can be processed per hour. This yields enough algal matter to be turned into value chemical products economically.97 The US DOE study from 1978 – 1996 concluded that the constant reseeding needed to avoid contamination and other factors listed above meant that raceway and circular designs were the much more effective for producing algae.94

2.1.2 TANKS

A similar design to the open pond is an artificially made tank, constructed outside. Conceptually similar to an open pond the much smaller volumes of biomass producible are offset by the increased control of environmental factors.93

2.1.3 CIRCULAR / RACEWAY PONDS

A circular system or raceway pond consists of a closed loop where the algal matter is recirculated around a channel. The mixing and circulation is achieved by a paddlewheel and baffles situated on the corners (Fig 2.1). As the mixing is mechanically operated the depth of the water can be reduced, 30 cm is normal. These channels are usually built in concrete or compacted earth and lined with plastic. As the sun is used as the light source, the algae is only fed during daylight hours, the nutrients enter the reactor just before the wheel while the algal matter is harvested from behind the wheel. The paddlewheel operates 24 hours a day, to prevent sedimentation.98

33 Chapter 2: Bioreactor Design

Fig. 2.1 General structure of a raceway pond taken from Christi et al.98

There are significant problems in the production of algae in using these systems. Temperature fluctuates with a diurnal cycle, as well as seasonally. Cooling is only achieved by evaporation, and evaporative water losses are significant. Open raceway ponds use CO2 much less efficiently than other type reactors, and despite bubbling

CO2 systems being used in NREL the maximum growth was still low. Despite the lower costs involved in the production of the algae, the concentration of biomass remains low as the mixing is comparatively inefficient and the reactors cannot sustain a dark zone.94, 98

2.1.4 SLOPED OUTDOOR REACTORS

The systems discussed above generally generate a maximum of 1 g m-2 d-1. By reducing the depth of the algae to 0.01 m and sloping a glass panel at an inclination of 1.7%, far better mixing and light absorbance meant that the reactor could sustain denser populations than those in raceway ponds, Energy cost relating to circulation and pumping were also reduced as were costs relating to the harvesting (due to the denser material). This reactor was still open to invasion by other algal strains and microscopic creatures, and as such had to be cleaned daily.99

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2.2 CLOSED REACTOR DESIGN

Because of the constraints discussed above open systems seem to have reached the upper limit of technological advancement, and there seems little room for improving yields beyond what has already been achieved. An alternative to the open pond system is to use a closed reactor. Among the many advantages are that these reactors are more appropriate for sensitive strains (including algae which does not grow in extreme environments, or when the products formed are susceptible to microbial degradation). As contamination is easily controlled photoautotrophic, heterotrophic or mixotrophic growing modes can all be used. Among other factors this means that cell density is increased massively and therefore the volumes needed, and harvesting costs are significantly reduced. However, high capital investment costs as well as high production costs mean that until recently no industrial sized closed reactors were used. One of the key improvements which have changed this is the improved efficiency of the parameters discussed in Chapter 1. There are several parameters that affect reactor design; these include light penetration (quality and quantity), a steady supply of nutrients, the gaseous transfer of CO2 into the reactor and O2 out of it, the efficient mixing of the algal broth as well as temperature and pH. There are a number of reactors of varying shapes which have been investigated previously, all of which have different advantages and disadvantages depending on utility. The three basic designs are tubular reactors, flat plate reactors and fermenter type reactors.100 The first two are generally transparent and use direct sunlight as the light source, the fermenter-type reactors tend to use either LED arrays or a mix of natural and artificial illumination.101

2.2.1 TUBULAR REACTORS

There are three general types of tubular reactor, a vertical reactor comprised of a 102 transparent tube where CO2 is bubbled in at the bottom, a horizontal reactor also made from transparent materials where a gas transfer system is used to exchange CO2 103 for O2, there a helical design where the transparent tube is coiled in some way to maximise the amount of light.104

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2.2.1.1 Vertical Tubular Reactors (VTR)

Vertical tubular reactors are made from transparent solid polyethylene or glass tubes, even though flexible polyethylene bags have also been widely experimented with. For all these VTR devices air is entered in the bottom, via either a bubble or airlift mechanism (Fig 2.2) and this supplies the necessary amount of CO2 while also 100, 102 keeping the level of O2 to a minimum. The advantage of using this type of reactor are the high surface to volume ratio, low shear forces, low cost and the absence of wall growth. Cleaning and sterilization, along with the inherent fragility of the tubes is a considerable disadvantage in this design.105

Fig. 2.2 Air lift and bubble column reactor designs, taken from Sevilla et al.106

Flexible polyethylene bags have been the most widely used due to their low cost and inherent sterility. These flexible bags can produce up to 3 times higher growth rates that the best open pond systems and reactors with a capacity of 20–50 L have been reported. However, the bags tend to be fragile while there are

36 Chapter 2: Bioreactor Design insurmountable problems on scaling the technology up to an appropriate industrial scale.107-110 The benefits of a bag reactor (without some of the draw backs) can be achieved by fixing a plastic film to a rigid framework.111 Chae et al. built a laboratory scale (100 L) photobioreactor to cultivate Euglena gracilis (a culture which typically likes acidic conditions and ambient temperatures). The reactor was equipped with a cover and baffles that induced plug-flow of the medium (Fig. 2.3 A, B). The broth was fed on CO2 and air. The light was provided by fluorescent lamps. This design was then used on a pilot scale (1000 L). This scaled up version was lit by sunlight and supplied with a steady source of flue gas (Fig. 2.3 C, D).112

Fig. 2.3 Schematic (A) and cross-sectional (B) diagrams of a laboratory scale photobioreactor for semi-continuous and contnous culture. Schematic (C) and Cross sectional photograph (D) of pilot-scale bag reactor, taken from Chae et al.112

Rigid VTR systems (made from plastics or glass) are more promising. The solid VTR are low cost and easy to operate and are generally made from a solid transparent tube, either lit from the inside by artificial lighting or from direct sunlight. The wall can be a double layer, with a refrigerant circulated to keep the optimum temperature under close control.105 Again, however, scale up is not straight forward. The reactor diameter must be relatively high to keep an efficient gas transfer and provide a large enough culture volume, but this in turn decreases the area-to-volume

37 Chapter 2: Bioreactor Design ratio and consequently constrains the photo efficiency. Also the vertical reactor is not set at the optimum angle to collect direct sunlight and a large amount of the energy is reflected back rather than being absorbed by the biomass.

2.2.1.2 Horizontal Tubular Reactors (HTR)

HTR systems do not possess this problem and have an angle to the sun which gives a much higher energy capture. With this added efficiency, and the closed nature of the system, the reactors can handle large volumes of algal matter. This exposure to the sun can be problematic in controlling the temperature and swings of up to 20 °C have been observed. Gas transfer can also be problematic with a dedicated gas-exchange 103 units mainly being used to deliver CO2 to the broth and keep O2 levels normal.

Fig. 2.4 Examples of horizontal reactor design, taken from various sources100, 113

There are many ways to control the temperature of HTR uints. Torzillo et al. built a HTR which covered 80 m2 and had a working volume of over 8000 L. This reactor was used for the production of Spirulina platensis, which was driven around the feeding tank, by a diaphragm pump. The reactor achieved a productivity of 0.25 g L-1 d-1 which did not increase on scale up mainly due to the wildly fluctuating temperatures. Four methods were examined; shading the tubes, overlapping the tubes, spraying the tubes with water and switching to a more temperature tolerant strain of algae. The last two were the most effective and were used in conjunction from then on.113

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Other HTR systems are mainly used conjunction with VTR units. Several reports have used a design layout where horizontal tubes are used for light harvesting connected to a gas exchange tower unit (Fig. 2.4 B). Algae are pumped around the system at between 30-50 cm s-1 which allows for low shear rates and good mixing. The horizontal tubes are made from plastic (polycarbonate or plexiglass) and the temperature controlled by either water spraying or submergence in a pool of water. Theoretically scale up is no problem as additional horizontal sections can be added to the VTR gas exchange section, this does have the inherent drawback of using a large amount of land or cooling water.114-116 A near horizontal tubular reactor has also been reported, with an angle of 5° to the horizontal plain. Again this system has efficient light gathering and a high area to volume ratio, yet poor temperature control was yielded when using a water spray (presumably as the water does not sit on the tubes absorbing the heat but rather runs off it) and poor gas transfer.117 A novel similar design to this one was described by Lee et al. In this they placed tubes in an α-shape, the algal broth was then pumped around by airlifts and nutrients injected at various places in the tubes. This makes the light harvesting efficient yet takes up vary little space.118

2.2.1.3 Helical Tubular Reactors (HETR)

The large land demands of horizontal reactors can be improved on by aligning the tubes in a helical pattern instead (Fig. 2.5). A gas exchange tower is placed at the top and the broth is driven around the tubes up to this unit by a centrifugal or airlift pump. These reactors have large area to volume ratios and a light source can be placed in the centre of the coil to compensate for the poor angle to the sun. Temperature control is also problematic and a dedicated heat exchanger invariably must be used. The last problem involved in this design is the strength that the algae need to be circulated this creates a large sheer stress and extensive cell damage. This is kept to a minimum if an airlift pump is used instead of a centrifugal pump. There is also some question about the ability to scale up this design. 93, 104, 119-121

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Fig. 2.5 A straight helical reactor described by Hall et al. 122 and a conical helical reactor described by Morita et al.121

2.2.2 FLAT PLATE REACTORS

An alternative to the tubular reactor, which can make the more efficient use of sunlight while retaining high area to volume ratios are flat plate reactors (FPR). These reactors (Fig. 2.6) tend to have high mixing rates coupled with low sheer stress. Oxygen build up is generally the largest problem which needs to be overcome and can be done so by an open air gas exchanger, however, this makes the system more susceptible to attack from external organisms. There are three basic designs, the first being a flat sheet (Fig 2.6 A) where gas can be bubbled in via perforated piping within the system, these panels can then be sprayed/submerged in water for cooling. The advantage of these is that any number of these units can be placed together to scale up. The efficiency of these sheets is on a par with the VTR technology.123 An improvement on this design is a V shaped flat plate reactor (Fig 2.6 B). This specific reactor described by Iqbal et al. has high mixing rates and very low shear stresses. This design has only been built on a laboratory scale.124 A more successful concept when scaling up are to use alveolar panels (Fig. 2.6 C).125, 126 These panels can be horizontally or vertically aligned while the mixture can be mixed and degassed by bubbling air through the bottom of each channel. These systems are extremely productive, with a uniform amount of light being distributed across the reactor. These reactors have also been scaled up successfully to industrial size, however oxygen build up is a large problem and generally these reactors are coupled to an open raceway pond to aid in the degassing. Even though this improves

40 Chapter 2: Bioreactor Design the productivity of the raceway, the algal strains become vulnerable to invasion from other organisms.

Fig. 2.6 Three flat plate reactor designs taken from Malcata et al.100

2.2.3 FERMENTER TYPE REACTORS

It is also possible to grow algae in fermenter type reactors (FTR). The area to volume ratio is very small, so using direct sunlight is highly inefficient. To overcome this most fermenter type reactors are illuminated from the inside by artificial lighting (normally fluorescent lamps) In these systems the mixing tends to be excellent while the sheer stress can also be kept to a minimum. As the reactors do not have to be stored outside, cross contamination is not an issue. The algae is normally fed by bubbling a CO2 enriched stream of air in through the bottom of the reactor, the operation parameters can also be strictly controlled to give the efficient production of the biomass.127

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Fig. 2.7 Fermenter type reactor described by Malcata et al. 100

More recently several advances in designs have been reported, however, it should be noted that no large scale processing exists in this type of reactor. Sevilla et al. reported on a computer controlled, 5-L reactor with a diameter of 170 cm. This reactor was sterilized with heat and the light was provided by 16 fluorescent lamps with cylindrical reflectors placed around the reactor. Nutrients, pH and temperature were all carefully controlled automatically. One of the main disadvantages in this type of system is the energy costs in the illumination.128, 129 Ogbonna et al. significantly reduced the use of artificial light by designing an integrated solar and artificial light system to provide internal illumination of a photobioreactor. In this system light is gathered using a number of solar collectors fixed on light tracking apparatus. The light is collected and fed into the reactor via optical fibres. At night, as well as during cloudy or rainy sections, a metal halide lamp fed light into the optical fibres to maintain illumination (Fig. 2.8).101

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Fig. 2.8 Designs for a light tracking and artificially lit fermenter type reactor, taken from Ogbonna et al. 101

Lamps emit light across the whole visual spectrum and in some case the UV and IR as well. This is inefficient and the overall energy conversion can be improved by using LED light sources. Lee et al. designed a photobioreactor system using LED emmiting at 680 nm as the sole light source. The LED units were located internally and emitted a photosynthetic photon flux of up to 50 mW cm-3. Cell concentrations of more than 2 x 109 cells/ml were observed with a total cell volume fraction of 6.6% v/v.130 Many other LED type reactors have been described in the literature using red or blue LED lights as the sole light source.131-136 Despite there being no industrial examples of algae produced in this manner, it should be noted that there is a vast experience in the pharmaceutical and food industries of scaling up this type of reactor for different uses, and a fermenter type reactor could become a competitive alternative to open pond technology.100

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2.3 PROCESSING

Most cases of algal manufacture require the algae to be separated from the water, harvested, dried and the relevant products extracted. The exception to this is when algae are being used for waste treatment or fish food cultivation.

2.3.1 SEPARATION OF MICROALGAE

All algae have a density which is close to water and a general negative charge across the surface of the cell. When the algal culture is young, and growth is still rapid the negative charge is high and the cells remain dispersed across the medium. After this stage is over, and the algal broth is old and has ceased to grow rapidly there is a decrease in the negative charge, organic polymers are secreted and the algal broth starts to coagulate (autoflocculation). The first stage of algae growth is desirable for the cost effective production of algae but the harvesting is both difficult and expensive. In the second scenario it is easier to harvest the biomass but the general productivity of the reactor is decreased due to the long residence time of the algae. A trade off has to be made where harvesting costs are least and productivity is greatest. Below are listed some of the different methods utilised in harvesting algal matter.137

2.3.1.1 Centrifugation

All algae can be concentrated using centrifugation. Continuous disk type centrifuges usually yield a stream with as ‘milk-like’ consistency containing 1-2% water free solids (WFS). For higher concentrated streams further techniques, detailed below, need to be used in conjunction with the centrifuge. However, solid bowl centrifuges, plate separator, nozzle and decanter technology can all be used as well to achieve more concentrated streams. All strains can be concentrated in this manner, and the final algal product will contain no additives however, centrifugation can be expensive and energy consuming as well as being damaging to more sensitive cells.138

2.3.1.2 Sedimentation and Flotation

To concentrate algae by sedimentation or flotation requires built for purpose tanks in addition to the reactor vessel. For sedimentation, tapering conical tanks are used

44 Chapter 2: Bioreactor Design where the algae can be tapped off. Use of a valve coupled with a pump to collect the sediment is the most common method.139 Neither of these techniques are effective for algae which does not autoflocculate unless flocculating agents are also used. The main agents, which are toxicologically safe (suitable for food production) are chitosans and vegetable starches, the latter being more widely available and inexpensive.138 If the algal matter is used for other purposes then aluminium or ferric sulphates (or chlorides) can be used instead.137 These reagents form hydroxides (in conjunction with calcium hydrogen carbonate) which are insoluble, clump together and entrap the algal cells. After the use of these agents the clumped together algal matter will either float or start to sediment depending on the density. This is reliant on the strain and techniques employed. However most algae will more readily float and flotation techniques tend to be more efficient and rapid than sedimentation, solid fractions of up to 7% have been reported. Flotation of trapped particles can be enhanced by releasing entrapped gases (air, O2, CO2) into the water or even hydrogen generated from electrolysis of the water.140 Generally algal matter will float upwards far easier than it will sediment. Floatation tanks are cheaper and easier to use than centrifugation and the algal matter is easier skimmed from the surface than collected at the bottom. Float matter has a lower water content than that collected by sedimentation. It should also be noted that certain algae will float and sink intermittently due to gas vacuoles and can be harvested without the use of flocculating agents.137 The main draw back of this technique is the amount of additives that are present in the final product, which then have to be removed with either an acid wash or some further industrial step.

2.3.1.3 Filtration

Algae can be removed simply from the aqueous media by vibrating, oscillating or cascade screens with openings of around 100 µm. This only applies to filamentous algae such as Spirulina and not to strains like Chlorella or Scenedesmus. These non- filament strains can be caught with screens with much smaller openings (5 µm) however this greatly reduces the flow rate of the screens, as well as the screens getting regularly blocked.137, 141, 142

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More advanced methods of filtration (such as filter presses, diaphragm presses rotary vacuum filters, vacuum band filters, sand bed filters etc.) have been investigated for their applicability in removing algae from water. All filtration devices suffer from rapid clogging and cleaning issues and the choice method is dependent on the algal source used.138 Microstraining, is probably the cheapest method of harvesting algae and does not involve additives at any stage however this method really is only applicable to filamentous strains.138

2.3.1.4 Phototactic Separation

Motile algal strains will react and move away or towards certain frequencies of light, even though there are large technical problems involved with the amount of light coverage and penetration it might be possible to harness this action to easily gather certain algal strains.137, 143

2.3.2 PROCESSING EXTRACTED MATTER

2.3.2.1 Drying

Once the algal broth has been thickened by the harvesting then it can be processed. The first method is drying, algae can be dried to improve the storage but on drying the algae will release ammonia and some organic acids as well as the water. Algae can be dried on a porous material like sand, by spray drying or in a continuous drum dryer or by spreading thinly on plastic sheets outdoors. The first technique is simple, yet some of the quality is lost and the sand will adhere to the surface of the dried biomass. This technique is usually only applicable when producing fertilisers or chicken feed. Similarly the algal mass can be left on black plastic sheets in the sun, though this is a remarkably efficient way of drying the matter, large land spaces are needed, precipitation can be problematic and only around 100g m-2 day-1 can be processed. More high-tech solutions are to use a drum dryer, though efficient and rapid, the algal broth is not very dense and the blades have to be sharp, the release of ammonia and organic acids also mean that special stainless steel technology has to be used. The most expensive method is to spray dry the algal matter. This technique is commonly used for Spirulina manufacture and produces a fine green powder that can be stored in

46 Chapter 2: Bioreactor Design the dark indefinitely. The pigments, chloro- and xanthophyll compounds or proteins can also then be extracted with ease with a non-polar solvent.137

Fig. 2.9 Algal matter drying process taken from Spolaore et al. 144

2.3.2.2 Extraction of Components by Degradation of the Cell Wall

To extract lipids or water soluble components from the algae the cell wall must be either broken down or disrupted. Solvents (such as hexane, methanol or scCO2) ultrasonicators and homogenisers are also used for this process. In certain species a salt imbalance can be enough to burst the wall osmotically, this can also be achieved by injecting CO2 under pressure. Once the cell wall has been broken the valuable components must then be separated from one another.145, 146 To extract edible protein mixtures and improve the digestibility of the algae, cooking at high temperatures, soaking in urea as well as disrupting the cell walls by ultrasonic and homogenizer technology have all been attempted.145

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2.4 REFERENCES

91. J. U. Grobbelaar, Journal of Applied Phycology, 2000, 12, 201-206. 92. M. A. Borowitzka, Journal of Applied Phycology, 1992, 4, 267-279. 93. M. A. Borowitzka, Journal of Biotechnology, 1999, 70, 313-321. 94. J. Sheehan, A Look Back at the U.S. Department of Energy's Aquatic Species Program - Biodiesel from Algae, NREL, Colorado, 1998. 95. D. Chaumont, Journal of Applied Phycology, 1993, 5, 593-604. 96. L. J. Borowitzka, Bioresource Technology, 1991, 38, 251-252. 97. L. Schlipalius, Bioresource Technology, 1991, 38, 241-243. 98. Y. Chisti, Biotechnology Advances, 2007, 25, 294-306. 99. J. Doucha and K. Livansky, Algological Studies, 1995, 76, 129-147. 100. A. P. Carvalho, L. A. Meireles and F. X. Malcata, Biotechnology Progress, 2006, 22, 1490-1506. 101. J. C. Ogbonna, T. Soejima and H. Tanaka, Journal of Biotechnology, 1999, 70, 289-297. 102. X. Zhang, B. C. Zhou, Y. P. Zhang, Z. L. Cai, W. Cong and O. Y. Fan, Biotechnology Letters, 2002, 24, 1767-1771. 103. B. Santek, M. Ivancic, P. Horvat, S. Novak and V. Maric, Chemical and Biochemical Engineering Quarterly, 2006, 20, 389-399. 104. M. Morita, Y. Watanabe, T. Okawa and H. Saiki, Biotechnology and Bioengineering, 2001, 74, 136-144. 105. K. Miyamoto, O. Wable and J. R. Benemann, Biotechnology Letters, 1988, 10, 703-708. 106. J. M. F. Sevilla, M. C. C. Garcia, A. S. Miron, E. Belarbi, F. G. Camacho and E. M. Grima, Biotechnology Progress, 2004, 20, 728-736. 107. F. Martinezjeronimo and F. Espinosachavez, Journal of Applied Phycology, 1994, 6, 423-425. 108. E. Cohen and S. M. Arad, Biomass, 1989, 18, 59-67. 109. I. Laing and E. Jones, Aquacultural Engineering, 1988, 7, 89-96. 110. P. Trotta, Aquaculture, 1981, 22, 383-387. 111. Italy Pat., PCT WO 2004/074423 A2, 2004.

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112. S. R. Chae, E. J. Hwang and H. S. Shin, Bioresource Technology, 2006, 97, 322-329. 113. G. Torzillo, B. Pushparaj, F. Bocci, W. Balloni, R. Materassi and G. Florenzano, Biomass, 1986, 11, 61-74. 114. E. M. Grima, J. A. S. Perez, F. G. Camacho, J. L. G. Sanchez, F. G. A. Fernandez and D. L. Alonso, Journal of Biotechnology, 1994, 37, 159-166. 115. A. S. Miron, A. C. Gomez, F. G. Camacho, E. M. Grima and Y. Chisti, Journal of Biotechnology, 1999, 70, 249-270. 116. A. Richmond, Hydrobiologia, 1987, 151, 117-121. 117. G. C. Zittelli, F. Lavista, A. Bastianini, L. Rodolfi, M. Vincenzini and M. R. Tredici, Journal of Biotechnology, 1999, 70, 299-312. 118. Y. K. Lee, S. Y. Ding, C. S. Low, Y. C. Chang, W. L. Forday and P. C. Chew, Journal of Applied Phycology, 1995, 7, 47-51. 119. M. A. Borowitzka, Journal of Marine Biotechnology, 1996, 4, 185-191. 120. European Pat., 261,872, 1988. 121. M. Morita, Y. Watanabe and H. Saiki, Biotechnology and Bioengineering, 2000, 69, 693-698. 122. D. O. Hall, F. G. A. Fernandez, E. C. Guerrero, K. K. Rao and E. M. Grima, Biotechnology and Bioengineering, 2003, 82, 62-73. 123. A. Richmond and Z. Cheng-Wu, Journal of Biotechnology, 2001, 85, 259-269. 124. M. Iqbal, D. Grey, F. Stepansarkissian and M. W. Fowler, Aquacultural Engineering, 1993, 12, 183-190. 125. M. R. Tredici, P. Carlozzi, G. C. Zittelli and R. Materassi, Bioresource Technology, 1991, 38, 153-159. 126. M. R. Tredici and R. Materassi, Journal of Applied Phycology, 1992, 4, 221- 231. 127. T. Stadler, Algal Biotechnology, Elsevier, New York, 1988. 128. E. M. Grima, E. G. Camacho, J. A. S. Perez, E. G. A. Fernandez and J. M. F. Sevilla, Journal of Applied Phycology, 1996, 8, 529-534. 129. E. M. Grima, J. A. S. Perez, F. G. Camacho, J. M. F. Sevilla and F. G. A. Fernandez, Applied Microbiology and Biotechnology, 1994, 41, 23-27. 130. C. G. Lee and B. O. Palsson, Biotechnology and Bioengineering, 1994, 44, 1161-1167.

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131. C. G. Lee and B. O. Palsson, Journal of Fermentation and Bioengineering, 1995, 79, 257-263. 132. H. C. P. Matthijs, H. Balke, U. M. VanHes, B. M. A. Kroon, L. R. Mur and R. A. Binot, Biotechnology and Bioengineering, 1996, 50, 98-107. 133. K. H. Park, D. I. Kim and C. G. Lee, Journal of Microbiology and Biotechnology, 2000, 10, 817-822. 134. Y. S. Yun and J. M. Park, Applied Microbiology and Biotechnology, 2001, 55, 765-770. 135. D. T. Nhut, T. Takamura, H. Watanabe, K. Okamoto and M. Tanaka, Plant Cell Tissue and Organ Culture, 2003, 73, 43-52. 136. S. J. Oh, D. I. Kim, T. Sajima, Y. Shimasaki, Y. Matsuyama, Y. Oshima, T. Honjo and H. S. Yang, Fisheries Science, 2008, 74, 137-145. 137. W. J. Oswald, in Micro-Algal Biotechnology ed. M. A. Borowitzka, University Press, Cambridge, Editon edn., 1988, pp. 357-394. 138. F. H. Mohn, in Micro-Algal Biotechnology, ed. M. A. Borowitzka, University Press, Cambridge, Editon edn., 1988, pp. 395-414. 139. F. H. Mohn, in Algae Biomass, ed. G. Schelef, Elsevier, Amsterdam, Editon edn., 1980, pp. 547-571. 140. E. Sandbank, G. Shelef and A. M. Wachs, Water Research, 1974, 8, 587-592. 141. R. A. Kormanik and J. B. Cravens, Water & Wastes Engineering, 1978, 15, 72- 74. 142. R. A. Kormanik and J. B. Cravens, Water & Sewage Works, 1979, 126, 31-35. 143. O. A. Sineshchekov, E. G. Govorunova, A. Der, L. Keszthelyi and W. Nultsch, Journal of Photochemistry and Photobiology B-Biology, 1992, 13, 119-134. 144. P. Spolaore, C. Joannis-Cassan, E. Duran and A. Isambert, Journal of Bioscience and Bioengineering, 2006, 101, 87-96. 145. W. J. Oswald, in Microalgal Biotechnology ed. M. A. Borowitzka, University Press, Cambrisge, Editon edn., 1988, pp. 357-394. 146. A. Ramirez Fajardo, L. Esteban Cerdan, A. Robles Medina, F. G. Acien Fernandez, P. A. Gonzalez Moreno and E. Molina Grima, European Journal of Lipid Science and Technology, 2007, 109, 120-126.

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CHAPTER 3

ALGAE DERIVED PRODUCTS AND TECHNOLOGIES

Algae show great metabolic diversity and a large number of commodity chemicals can be isolated from algal matter. A diverse range of products can be isolated, including commodity chemicals (pharmaceuticals, nutraceuticals and pigments) and bioderived fuels (hydrogen, biodiesel, bioalcohols and methane).

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3.1 PHARMACEUTICAL PRODUCTS

Natural products offer a consistent source of new drugs and biomolecules, that possess a great level of diversity that is underrepresented in most synthetic compounds. Some of the key properties which are advantageous in chemicals produced by living organisms are the water solubility, cell membrane permeability and bioavailability.147 All difficult properties to engineer in the laboratory.148 Various compounds (either drugs or their precursors) which are currently being researched in this field are listed below by their drug class, the term antibiotic is taken to mean a substance which is either antifungal, antibacterial or antiprotozoal and so has been divided under these headings accordingly, the major compound structures are listed in Appendix I.

3.1.1 ANTHELMINTIC

This class of drugs expel parasitic worms. Currently relatively simple cyclic amines are in use medically, such as piperazine or albendazole related compounds. Seven brominated diterpenes of the parguerene and isoparguerene series were isolated from the red algae Jania rubens (L.) Lamx and were shown to have a significantly higher effect than the standard mebendazole (an active compound in various antithelmintic drugs currently on the market).149

3.1.2 ANTIBACTERIAL

There are many biomolecule compounds which have antibacterial properties. Algal extracts have been used previously as antibacterial agents. Chlorella vulgaris and Chlamydonas pyrenoidosa, have both been shown to have in vitro antibacterial properties.150 Another green algae extract, Ulva lactuca, has showed marked resistantance to the bacterium methicillin-resistant Staphylococcus aureus (MRSA).151 Cyanobacteria have also got antibacterial agents and the aqueous extracts of Nostoc muscorum and Synechocystis leopoliensis have both been demonstrated to have antibacterial effects,152 as has Aphanizomenon flos-aquae.153 Extracts of Mastocarpus stellatus, Laminaria digitata and Ceramium rubrum (all brown algae species) have also been shown to exhibit antibacterial properties.154

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Soil samples containing different species of green algae and yellow-green algae were also found to contain antibacterial compounds.155 Until recently relatively little research has been invested in determining the exact molecules which possess these properties and isolating them for further pharmaceutical use (Table 3.1).

Compound Algal Type Algae Studied Reference Bromophenol Red algae Rhodomela Confervoides 156, 157 Poly Unsaturated Diatoms Examples from most genera 158 Aldehydes (PUA) Hormothamnin A Hormothamnion Cyanobacteria 159 (peptide) enteromorphoides Kawaguchipeptin B Cyanobacteria Microcystis aeruginosa 159 (peptide) Norharmane (and Cyanobacteria Nostoc sp. 159, 160 derivatives) Ionones, phytols* Green algae Dunaliella sp. 161 Bromoform, Dibromo Red algae Asparagopsis armata 162 acetic acid Sulphoglycerolipid Brown algae Sargassum wightii Greville 163 Parsiguine Cyanobacteria Fischerella ambigua 164 Eucheuma serra, Galaxaura Lectin† Red Algae 165 marginata α-dimorphecolic acid, coriolic acid Oscillatoria redekei Cyanobacteria 166 (hydroxy unsaturated Limnothrix redekei HUB 051 fatty acids) Table 3.1 Examples of isolated biomolecules from various algal classes which display antibacterial properties

* These are decomposition products of chlorophyll and other carotenes, they are likely to be found in other classes of algae † Lectins are proteins which can bind specific sugars, many algal types other than red algae produce them. Lectins are highly specific antibacterial agents and must be matched to the correct bacteria.

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3.1.3 ANTICANCER

Though less extensively researched than antibacterial agents, algae also produce compounds with marked anticancer activity. Given below are the general groups of compounds which can act as interesting drug precursors and as potential anticancer drugs.

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Compound Algal Type Algae Studied Reference Terpenoid/ Generally most prevalent in Most 167, 168 isoprenoid lipids* macroalgae Acetylenic Marine based organisms 169-171 metabolites Allenic / Cumulenic Brown algae 172 fatty acids Allenic / cumuleneic brominated complex Red Algae Laurencia 172 lipids Most genera (however related Brown algae / compounds are found in Fucoxanthins 172 Diatoms almost all marine algae classes) Polysaccharide† Green algae Chlorella pyrenoidosa 173 Callophycoic Acids Red algae Callophycus Serratus 174 and Callophycols Plastocyanin derived Cyanobacteria Several genera 175 peptides & Green algae Canthaxathin (type of Chlorella (found in Green algae 176 tepernoid) cyanobacteria also) Fucoidan (sulphated Brown Algae Cladosiphon okamuranus 177 polysaccharide) Table 3.2 Plausible precursors to anti cancer drugs found in algae

Some biomolecules which can be derived from algae have been used in preclinical trials, these compounds are listed overleaf in Table 3.3.

* Terpenoids are important precursors for a large number of drugs but thought of as especially important in cancer research. † Specific polysaccharides weighing 69658 Da and 109406 Da respectively and being comprised of rhamnose.. mannose, glucose, galactose and an unknown sugar.

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Compound Chemical Class Molecular Target Source Organism Dolastatin / Cyanobacteria Linear Peptide Tubulin Synthadotin Symploca sp. Cyclic Green algae Kahalalide F Lysosomes depsipeptide Bryopsis sp. Dehydrodldemnin B Cyclic Ornithine Cyanobacteria (Aplidine) depsipeptide decarboxylase (possibly) Cyanobacteria Cemadotin Linear Peptide Tubulin Symploca sp. Cyanobacteria Soblidotin Linear Peptide Tubulin Symploca sp. Cyanobacteria Curacin A Thiazole lipid Tubulin Lyngbya majuscula Cyclic Cyanobacteria DMMC Tubulin depsipeptide Lyngbya majuscula Green Algae Caulerpenyne Sesquiterpene Tubulin Caulerpa sp. Any lipid Palmitic Acid Fatty Acid Apoptosis containing alga Amphidinolide (H2- Macrolide ? Dinoflagellates 5, G2-3) ma’iliohydrin Sesquiterpene ? Red Algae Table 3.3 Algae derived biomolecules which are in preclinical cancer trials, taken from Simmons et al. and Mayer et al.178, 179

3.1.4 ANTICOAGULANT

Anticoagulants are molecules which stop blood clotting. The main drug used presently for this purpose in the UK is Warfarin. Most algae will produce polysaccharides, of which the water soluble sulphated polymers have been shown to be effective anticoagulant biomoleules.180-183 These polysaccharides tend to occur in marine species, examples of which are found in almost all the algal divisions.

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3.1.5 ANTIFUNGAL

Generally the compounds listed as antibacterial (Section 3.1.2) also display antifungal properties. More specifically extracts of cyanobacteria have been screened for the antifungal properties and were found to be effective, especially the lipid soluble compounds.184 What specific biomolecules were active was not investigated but certain terpenes and norharmanes are known to inhibit fungal growth.185, 186 There is also evidence that hydrolytic enzymes can play a role in halting fungal growth.187

3.1.6 ANTIPLATELET

An antiplatelet drug decreases platelet (cells circulating in the blood) formation, they are therefore effective in arterial circulation where anticoagulants cannot be used. Aspirin is probably the most commonly used antiplatelet drug. Several compounds isolated from algae can also be used. Sulphated polysaccharides, derived from most classes of algae have been shown to be effective,188 as have some carrageenans.189

3.1.7 ANTIPROTOZOAL

Some compounds produced by algae are toxic to other algal species as well as some protozoa. Prodigiosin, a pigment found in brown algae is one of these compounds and can be used to halt harmful algal blooms.190 Other molecules also disrupt the blooming process. For example the aqueous fraction of three marine macroalgae species (Olva linza, Corallina pilulifera and Sargassum thunbergii) were shown to be powerful algicidal agents when tested on the red tide microalgae Prorocentrum donghaiense.191 Malaria, probably the most common infectious disease in the world, is caused by protozoal parasites. As the new strains of malaria are becoming resistant to the commonly used drugs such as chloroquine and hydroxychloroquine as well as quinine, novel drugs and precursors are being sort.192 Brominated sesquiterpenes from the red alga Laurencia obtuse have been shown to have antimalarial properties as have bromoditerpenes from the red alga Sphaerococcus coronopifolius.193, 194 A range of other diterpenes, isolated from brown alga, have also shown promise in this area.195

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3.1.8 ANTIVIRAL

Algae, and a number of biomolecules derived from algae, have long been used as antiviral medicines.196 Indeed in 2003 researchers pooling together a number of epidemiologic studies of HIV/AIDS concluded that a strong unifying characteristic of countries with anomalously low rates was the significant consumption as part of the diet.197 It has been suggested previously that rates of infection cannot solely be attributed to differences in IV drug use and sexual behaviour,198 and though there is unlikely to be one complete answer a number of compounds from certain algal species have been isolated and clinically proven to inhibit HIV and other viruses (Table 3.4).199

Compound Algal Type Algal Species Virus Ref. Norharmane Cyanobacteria HIV 200 Fucan polysaccharide Brown algae HIV 201, 202 Alginic acid Cyanovirin-N* Cyanobacteria Nostoc ellipsosporum HIV 203, 204 Microcystis viridis* Cyanobacteria Microcystis viridis HIV 203 203, 205, Griffithsin* Red Algae Griffithsia sp. HIV 206 Oscillatoria* Cyanobacteria Oscillatoria agardhii HIV 203, 207 agardhii agglutinin Scytovirin* Cyanobacteria Scytonema varium HIV 203, 208 Eucheuma serra * Red algae Eucheuma serra HIV 209 Herpes I Rhamnan sulphate Monostroma Green Algae HIV 210 (polysaccharide) latissimum HCMV Fucoidan† Brown algae Undaria pinnatifida Herpes I 211 Naviculan * Diatom Navicula directa Herpes I,II 212

* These compounds are all algal lectins, it is common practice to name the specific lectin after the algal species it is found in † sulphated polysaccharide

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Compound Algal Type Algal Species Virus Ref. Influenza A HIV Grateloupia filicina Sulphated Red algae Grateloupia HIV 213 polysaccaharides* longifolia Dolabelladienetriol Brown algae Dictyota pfaffii HIV 214 Chlorophyll c2 Brown algae Eisenia bicyclis IHNV† 215 (- Mg2+) Plastoquinones‡ Brown algae many 216, 217 Carrageenans Red algae Meristiella gelidium Herpes 218 isopachydictyolal Dictyota dicltotoma Herpes, 4α-acetyldictyodial Brown algae 219 D. linearis polio (Diterpenes) Pheophorbide Lyngbya sp. (chlorophyll related Green algae Herpes 220 Dunaliella sp. compounds) Some green Ceramides Encephalitis 221, 222 algae 223 Indolocarbazole Cyanobacteria Nostoc sphaericum Herpes Peyssonol A and B Red algae Peyssonnelia sp. HIV 224 Table 3.4 List of antiviral active drugs and drug precursors isolated from various algal species

* Sulphated polysaccharides have been extensively researched for their activity against many viruses and certain types show no antiviral properties at all, it is specific to type, shape and virus † Infectious Hematopoietic Necrosis Virus ‡ also precursors to antiviral chromene molecules

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3.2 FINE CHEMICAL PRODUCTS

3.2.1 VITAMINS

Algae like almost all higher plants produce and require vitamins. Vitamins are either extracted from the environment or synthesised in the cell. In some cases algae will excrete vitamins into the surrounding environment.150 Not all vitamins can be synthesised by mammals and a large amount need to be taken up as part of the diet. Vitamins are classified according to function and so most vitamins are actually groups of related compounds (Table 3.5). The term vitamin must not be confused with nutrients such as minerals, amino acids and fatty acids though these are also a necessary part of any diet. All though all vitamins can be found in our diet, the market for vitamin supplements is worth billions of dollars worldwide.225 All vitamins can be synthesised and collected from algal strains though information is fairly scarce on specific vitamin compounds (Table 3.6) The vitamin content of algae depends on the genotype, growing conditions, light intensity but perhaps most importantly the metabolism and the stage in the growth cycle that the algae is in. The vitamin content is therefore amenable to genetic manipulation to increase yields. For example a mutagen of Chlamydomonas eugamentos was grown to excrete 8 times more nicotinc acid into water than the original algal strain.226 The vitamin content of algae strains and the amount of vitamins available is comparable with bacteria and yeasts. Most of the screened algae presented in Table 3.6 are freshwater variants, this however is only due to historical factors and marine algae will also produce vitamins. Open pond technology is not desirable for vitamin synthesis as competing yeasts, bacteria and other algal strains will soon use the excreted vitamins and contaminate the culture. As with most of the chemicals produced, harvesting is an important consideration and for that reason it is important to examine whether the algae secretes the vitamins into the medium (on death of the cell or osmotically) as opposed to the vitamins being needed to be extracted.150 It should also be noted that evidence has been presented to suggest that some vitamin supplements (pre-vitamin A) have adverse health effects which would substantially decrease any future market share.227

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Vitamin Chemical Name Solubility Function Vitamin A Retinoids Fat Metabolism, Carotenes Tissue growth, Cell signalling

Vitamin B1 Thiamine Water

Vitamin B2 Riboflavin Water

Vitamin B3 Niacin Water Metabolism,

Vitamin B5 Pantothenic acid Water Immune system,

Vitamin B6 Pyridoxine Water Cell growth,

Vitamin B7 Biotin Water Chemical synthesis

Vitamin B9 Folic Acid Water

Vitamin B12 Cobalamin Water

Vitamin C Ascorbic Acid Water Antioxidant, enzymatic cofactor Vitamin D Cholecalciferol Fat Promotes Immune Ergocalciferol system and regulates blood Vitamin E Tocopherol Fat Antioxidant Tocotrienols Vitamin K Phylloquinone Fat Carboxylation of Menaquinone Glutamate residues Table 3.5 Vitamin types and function in the human body

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Alga B12 B6 B1 B7 B9 B2 B3 B5 C E A Cyanophyceae Anaebaena cylindrica 0.63-1.1 7 - R 15 55 77 88 2000 4000 - A. flos-aquae 0.28 ------A. variabilis ------1460 - Calothrix parietina 0.64 ------Chlorogloea fritschii ------1420 - Diplocystis aeruginosa 0.24 ------Gloeocapsa sp. ------1040 - Nostoc muscorum ------980 - Plectonema nostocurum 0.06-0.07 ------Spirulina maxima 2.0 3.0 55 - 0.5 40 118 11 - 190 - S. platensis 1.2-2.5 5-7 30-40 - 5000 25-35 105 5-8 - 50-70 - Anabaena hassali* Microcystis pulverana 1.99- 0.41-6.65 1.1-1.76 0.58 - 20-60 30.6-73.5 22-55.5 - - - Nostoc punctiforme 12.36 Phormidium bijugatum

Chlorophyceae Chlamydomonas eugametos ------119-129 - - - - C. eugametos (mutant) ------77-80 - - - -

* Also includes the species A. variabilis, Calothrix braunii, Nostoc sp., Calothrix sp., Phormidium autumnale

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Alga B12 B6 B1 B7 B9 B2 B3 B5 C E A C. reinhardtii 0.26 9 - - - 2000 4000 105 Chlorella ellipsoidea 0.04-0.09 ------Chlorella pyrenoidosa 0.02-0.1 23 10-41 0.14-2.5 6.4-28.3 27-80 120-240 3.2-20 - - - Chlorella vulgaris 0.06-0.07 11 - 0.49-1.44 11 68 56-247 79-182 15000 2000 - Coccomyxa sp. - - - 1.88 ------Eugelena gracilis R 10.3 - 0.15 8.8 39 30 37.7 - - - Scenedesmus acutus 1.4 - 63.8 0.8 3 150 26.2 44 760 526 - S. obliquus 0.4 - 17 0.2 6-7 40-46 120 15 2000 140-1000 - Chrysophyceae Poterioochromonas stipitata R 3.4 - R 6.8 19.3 46.7 18.3 1800 - - Ochromonas danica 23 R R 9.0 35 89 37 830 2170 137

Bacillariophyceae Chaetoceros simplex 0.047 1.84 3.15 1.75 2.1 5.3 62.3 29.5 - - -

Dinophyceae Peridinium cinctum 0.2 1-3 2-9 0.2-0.3 0.4-0.7 26.6 9-18 7 - - - Table 3.6 Vitamin contents of various algal species (µg (g dry weight)-1. R = Required for growth, - = no data available, adapted from Borowitzka et al. and references found within150

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3.2.2 PIGMENTS

The major pigment in algae is chlorophyll (a, b c1, c2 and d) yet there are also a vast range of other pigments such as phycobiliproteins (water-soluble proteins that capture light energy) and a wide range of carotenoids. Generally algae usually possess more than one pigment and these can be found in abundance.

3.2.2.1 Phycobiliprotein

Phycobiliproteins are present in most algae species and are essential in transferring light energy to chlorophyll. There are two main types; phycoerythrin and phycocyanin. R-phycoerythrin and B-phycoerythrin occur mainly in Rhodophyceae while C-phycoerythrin occurs in the Cyanobacteria. These pigments absorb blue/green radiation and emit orange/yellow light, however when other algae are present these pigments can appear bright red. R-Phycocyanin is found in Rhodophyceae while C-Phycocyanin and allophycocyanin occur in the Cyanobacteria. These pigments absorb orange/red light and emit blue radiation. The proteins are all water soluble and are harmless, which make them suitable as edible dyes for the food and additive industries, phycocyanin is extracted from Spirulina platensis commercially for this purpose. 150, 228

3.2.2.2 Carotenoids

Carotenoids are primarily used as edible dyes to either colour food or enhance certain colours such as salmon flesh, egg yolk or orange juice, even though some (like β- carotene) are sold as precursors to vitamin A. Carotenoids are generally yellow to red and function primarily as photoprotection agents for chlorophyll.150, 229, 230 There are over 400 known carotenoids but only a few are produced on a large scale, these are β-carotene, astaxanthin, lutein, zeaxanthin, lycopene and bixin (Table 3.7).231, 232 Some studies have suggested that carotenoids have anti-inflammatory and anti-cancer properties also. Carotenoids are mainly produced synthetically (usually as the trans form) as the naturally derived carotenes are expensive.233 The highest produced carotenoid is β-carotene, this compound is produced by D. salina and can be present in up to 14% of the dry weight. This algal species thrives under high salt and

64 Chapter 3: Products and Technology light conditions making it highly suited to outdoor cultivation. β-carotene usually retails for between $300-3000 kg-1.234 The next highest produced compound is astaxanthin and is used mainly as a pigment in aquaculture. The market for this compound has been estimated at $200 million with an average price of $2500 kg-1, however, around 90% of astaxanthin sold is produced synthetically by BASF and Hoffman-La Roche.148 Natural astaxanthin is produced by H. pluvialis in a 3% (dry weight) yield under low nitrogen and intense light conditions. The synthetic form is currently cheaper yet the natural astaxanthin is preferred in some industries as there is an enhanced deposition in cells and has the potential to be marketed as a natural product for human consumption.235 There are a few general environmental factors which can increase the production of carotenes. Generally depriving the algae of nitrogen, phosphate, iron and sulphate increases the yield. Growing the algae heterotrophically can also increase production. Glucose, glycerol and other organic substrates having been shown to be suitable.* An increase in light intensity has been shown to improve yields further. An increase in light intensity also generally widens the range of carotenoids produced. Temperature is not such a factor though D. salina does increase production at higher temperatures. pH has also been shown to effect the carotenoid content with individual algae species preferring one pH over others. It should be noted that carotenoid content does vary substantially depending on the taxonomy and the stage in the life cycle that the algae is in.150

* Whether the β-carotene percentage content was increased or the algae just had a faster growth rate is unclear.

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ae ae ycea ae e ycea phy yce o yceae yceae Carotenoid h hyce p hph ycea rioph ph phyce op ophyceae phyce do o n o ph en ho t dop rysoph cilla phi gl an no i y C Rho Cryptophyce D Ra Haptophyceae Ba Xan Eustigmat Eu Chlor Prasi Ch Astaxanthin - - - m - - - - - p - - - β-Carotene M M M m M M M M M M M M M α-Carotene - M M m - m - - - - M m - ε-Carotene - - m ------m - - Canthaxanthin m - - - - m - - p p (m) - - Cryptoxanthin m m - - - m - m - - - - - Diadinoxanthin - - - m - m M M - M - - - Diatoxanthin - - - m - m M M - m - - - Dinoxanthin - - - m ------Echinone m ------p (m) - - Fucoxanthin* - m - (M) - M M - - - - - M Heteroxanthin ------p - m - - - Lutein - M ------M m - Lycopene m ------Myxoxanthophyll M ------Neoxanthin ------(p) m - m m m p Oscillaxanthin M ------Peridinin ------Phytofloene - - - M ------Violaxanthin - m - m - - - - M - m m m Zeaxanthin m M ------(m) - M m m Table 3.7 Major carotenoids found in algal classes, M = major carotenoid, m = minor Carotenoid, p = present, () = in some species only, adapted from Borowitzka et al. and references found within150

3.2.2.3 Chlorophyll and Related Chemicals

There are five types of chlorophyll (a, b, c1, c2 and d) all five are green pigments as they absorb strongly in the blue and red regions. Algae always contain chlorophyll a

* A great range of these cartenoids are found in marine algae, an extensive review was conducted by Dembitsky et al. 172

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and then will contain one other depending on which class they belong to. Related compounds extractable from algae include phytol a suitable precursor to vitamins A, E and K.

3.2.3 AMINO ACIDS

There is a large market for free amino acids, the largest used are glutamic acid (> 300,000 t a-1 for human consumption), methionine (supplement for human and animals), lysine (essential amino acid used as an additive in animal feed), tryptophan (supplement and ‘natural’ pharmaceutical agent), aspartic acid (non-essential, used in synthesis of other amino acids) and phenylalanine (additive). Most amino acids are generally found in algal protein biomass except cysteine and methionine.150 Some algae excrete free amino acids into the environment during both the stationary and exponential growth phases. The identity and concentration of amino acids are all highly dependent on the strain as well as the position in the growth cycle of that particular species (Table 3.8).236 Generally though, high nitrogen conditions, high salinities and low levels of light will increase the production.237 Even though some bacteria can produce R- amino acids, algae generally produce the L- isomer.

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phyce ph o rioph ceros deb ceros a o o rella no t t rysoph i ctylum e e lo cilla D a a Haptophyceae Chlor Ch Ch oda Ba Ch Ch e Pha

Alanine P P m m m m m Arginine P m m (M) m m Asparagine P m m m Aspartic acid P P m m (M) m m Glutamic acid P m (M) (m) M M Glutamine P (M) m m Glycine P P m (M) M m m Histidine P Iso-leucine P Leucine P P M M m M Lysine P M (M) M m M Ornithine * P P Phenylalanine P m m (m) m m Serine P P P M (M) m m m Threonine P P m m m Tyrosine P m m m m m Valine P m (m) M m M Proline M Reference 238, 239 236 237 240 240 240 240 240

Table 3.8 Generalised summary of the amino acids excreted by algae. P = present, M= major product, m = minor product, brackets indicate variability depending on species, as this is a generalised summary it should be taken as a guide only, some amino acids are not present or were simply not examined

* Product in the enzymatic break down of arginine

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3.3 LIPIDS

Lipids are constituents of all plant cells. They have a wide range of functions including membrane components, storage products, metabolites as well as energy storage. They can be extracted using lipophillic (hydrophobic) solvents such as chloroform, ether or hexane. The major lipids can then be split into polar and non polar fractions. The non polar fraction is mainly made up of triglycerides where the polar fraction are glycerides with one or more fatty acid chains replaced by a polar group (i.e. phospholipids, glycolipids).241 This fraction also includes a number of the compounds reviewed above. There is a tremendous diversity across algal species but generally the lipid fraction ranges between 1-40 wt% with the triglyceride content constituting around 80% of that number* (Table 3.9 and 3.10).

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ae (eu ae (bl ycea yceae (g ho phyce t ae ( phyce o hyce o rioph Haptophyceae od phyce Cryptophyce yce (yellow-gree Xan op o h rysoph en p Rh an o cilla Chlor gl Ch y n C Ba Eu Di Total Lipids 2-23 12-72 5-48 3-17 6-16 5-36 1-39 1-70 17 (% dry weight) Neutral Lipids 11-68 44 41-58 14-60 21-66 (% total lipids) Glycolipids 12-41 17 42-59 13-44 6-62 (% total lipids) Phospholipids 16-50 39 10-47 17-53 (% total lipids) Hydrocarbons 0..1-1 trace 2.8 trace 0.5 (% dry weight) (39†) Table 3.9 Lipid distribution according to algal class, adapted from Borowitzka et al.242

* In comparison most vegetable oils contain 98% triglycerides † 39% belongs to Botryococcus braunii

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Lipid Content Algal Type Species Notes (% dry wt) Thalassiosira Brown Algae 21-31 pseudonana Botrydium 15 granulatium Diatoms Biddulphia aurita 40 Cyclotella DI-35 42 Cylindrotheca 28 High phospholipid fusiformis Hanzchia DI-160 66 Navicula pellisulsa 45 Nitzchia palea 40 High EPA Phaeodactylum 31 High EPA tricomutum Dinoflagellates Peridinium cinctum 36 Eustigmatophytes Nannochloropsis 31-68 Golden Algae Ochromonas danniea 39-71 Green Algae Ankistrodesmus TR-87 28-40 Botryococcus barunii 29-75 Hydrocarbons Chlorella 15-55 Heterotrophic protothecoides Chlorella pyrenoidosa 36 Chlorella sp. 29 Chlorella vulgaris 40-58 Chlorococcum 22 oleofaciens Dunaliella primolecta 54 marine Dunaliella salina 47 Dunaliella tertiolecta 36-42 marine Monalanthus salina 72 Nannochloris 6-63 Neochloris 35-54

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Lipid Content Algal Type Species Notes (% dry wt) oleobundans Radiosphara 43 negevensis Scenedesmus sp. 25 Scenedesmus TR-84 45 Stichococcus 9-59 Stichococcus 32 bacillaris Tetraselmis suecica 15-32 Haptophytes Chrysochromulina sp. 33-48 Isochrysis sp. 7-33 Table 3.10 Lipid distribution for various known species taken from Scragg et al.243

3.3.1 GENERAL FATTY ACID CONTENT

Even though there are Free Fatty Acids (FFA) present in some species of algae, the fatty acid content or fatty acid profile generally describes the amount of bound fatty acids. The triglyceride content of algal oils closely resembles that of vegetable oils yet the range of fatty acids is substantially higher in algae. These include fatty acids with an odd number of carbons as well as long chain polyunsaturated fatty acids (PUFA) not found in the higher plants. Lipid metabolism in algae, particularly the biosynthetic pathways of fatty acids and triglycerides, has been poorly studied. However, as some shared biochemical characteristics of a number of genes and enzymes isolated from algae are similar to the higher plants the general mechanisms are thought to be similar. 244, 245 In algae the de novo synthesis of fatty acids occurs in the chloroplast, a general scheme is given in Fig 3.1. The triglyceride synthesis has been proposed to occur by the direct glycerol pathway (Fig. 3.2). The exact mechanisms of these pathways and the metabolism of algae has been extensively reviewed by Hu et al. and more precise biological investigations can be found in this paper and references given within.245

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Fig. 3.1 Fatty Acid de novo synthesis pathway in chloroplast. Acetyl CoA enters the pathway as a substrate for acetyl CoA carboxylase (Reaction 1) as well as a substrate for the initial condensation reaction (Reaction 3). Reaction 2, which is catalyzed by malonyl CoA:ACP transferase and transfers malonyl from CoA to form malonyl ACP. Malonyl ACP is the carbon donor for subsequent elongation reactions. After subsequent condensations, the 3-ketoacyl ACP product is reduced (Reaction 4), dehydrated (Reaction 5) and reduced again (Reaction 6), by 3-ketoacyl ACP reductase, 3-hydroxyacyl ACP dehydrase and enoyl ACP reductase, respectively. Taken from Hu et al.245

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Fig. 3.2 Simplified schematic showing the triacylglycerol biosynthesis pathway in algae. (1) Cytosolic glycerol-3-phosphate acyl transferase, (2) lyso-phosphatidic acid acyl transferase, (3) phosphatidic acid phosphatase, and (4) diacylglycerol acyl transferase, taken from Hu et al.245

The triglyceride fraction of the lipid layer can be substantial and increased further when the algae are placed under adverse environmental conditions. Of the nutrients nitrogen is the largest limiting factor, however silicon, a sugar carbon source and phosphorus also can effect the lipid concentration and fatty acid profile. Temperature is a large factor in the general lipid concentration and though it has not been studied extensively, lower temperatures tend to increase levels of unsaturated fatty acids.245, 246 Light intensity is also important and can extensively change the lipid profile, generally lower light levels give a higher yield of polar lipids.247 Theoretically the glycerides in the algae could be used to produce biodiesel or other liquid fuels, however no commercially viable system has yet to be produced and triglyceride biotechnology focuses mainly on using algal oil as high-value nutritional source of PUFA. The various fatty acids that are of interest for this use are β-linoleic acid, γ- linoleic acid, α-linoleic acid, arachidonic acid, eicosapenaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid. It should be noted that these fatty acids can all be found in fish oils (mainly as algae in the plankton are the very basis of the marine food chain). Presently fish oil such as cod liver or salmon oils are much cheaper. The fatty acids present in algae, along with the abbreviations are given below. All fatty acids have nominal names, IUPAC names and also nomenclature to describe the number of carbons and double bonds in the chain. For example α-linolenic acid has eighteen carbons and three double bonds. This compound can be called 6, 9, 12- octadecatrienoic acid, α-linolenic acid or C18(3). As double bonds on the fatty acid

73 Chapter 3: Products and Technology chains are always three atoms apart (as well as being cis) there is no need to describe where all three are situated but rather to describe the bond nearest the end of the chain, this is termed the omega number. For α-linolenic acid the nomenclature C18(3) ω-3 is used (Fig. 3.3).

Fig. 3.3. Triglyceride with three fatty acid arms

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IUPAC Name Common Name (ω) Shorthand* Octanoic acid Caprylic C8(0) Decanoic acid Capric C10(0) Dodecanoic acid Lauric acid C12(0) Tetradecanoic acid Myristic acid C14(0) Pentadeconoic acid C15(0) Hexadecanoic acid Palmitic acid C16(0) cis-9-Hexadecenoic acid Palmitoleic acid (ω-7) C16(1) cis-6,9,12,15-Hexadecatetraenic acid ω-1 C16(4) Heptadecanoic acid C17(0) cis-10-Heptadecenoic acid ω-7 C17(1) Octadecanoic acid Stearic acid C18(0) Oleic acid cis-9-Octadecenoic acid C18(1) (elainic acid) (ω-9) cis-9,12-Octadecadienoic acid Linoleic acid (ω-6) C18(2) cis-9,12,15-octadecatrienoic acid α-linolenic acid (ω-3) C18(3) cis -6,9,12-Octadecatrienoic acid γ-linolenic acid (ω-6) C18(3) cis-6,9,12,15,-octadecatetraenoic acid Stearidonic acid (ω-3) C18(4) cis-11-eicosenoic acid Eicosenoic acid (ω-9) C20(1) cis-11,14-eicosadienoic acid Eicosadienoic acid (ω-6) C20(2) Dihomo-γ-linolenic acid cis-8,11,14-eicosatrienoic acid C20(3) (ω-6) (DGLA) Eicosanotrienoic acid (ω-3) cis-11,14,17-eicosatrienoic acid C20(3) (ETE) cis-5,8,11,14-eicosatetraenoic acid Arachidonic acid (ω-6) (AA) C20(4) Eicosatetraenoic acid (ω-3) cis-8,11,14,17-eicosatetraenoic acid C20(4) (ETA) Eicosapentaenoic acid cis-5,8,11,14,17-eicosapentaenoic acid C20(5) (EPA) (ω-3) Docosanoic acid Behenic acid C22(0) cis-13-docosenoic acid Erucic acid (ω-9) C22(1) cis-4,7,10,13,16-docosapentaenoic acid Osbond acid (ω-6) C22(5) Docosapentaenoic acid cis-7,10,13,16,19-docosapentaenoic acid C22(5) (ω-3) (DPA) Docosahexaenoic acid (DHA) cis-4,7,10,13,16,19-docosahexaenoic acid C22(6) (ω-3) Table 3.11 List of fatty acids commonly found in algae

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a e gree

l i l o n) g e- agel ae l u (green) e (d of ae (red yceae n ae i ph d

ae (eu ae (bl ycea yceae (g ho phyce t ae ( phyce o hyce o rioph Haptophyceae od phyce Cryptophyce yce (yellow-gree Xan op o h rysoph en p Rh an o cilla Chlor gl Ch y n C Ba Eu Di 14(0) 40 20 10 20 20 10 20 20 10 20 16(0) 60 20 20 30 30 30 40 30 30 20 16(1) ω-7 50 30 30 20 40 10 30 50 20 10 16(2) ω-7 20 10 10 tr. tr. - 10 10 10 10 16(2) ω-4 [50] 10 10 10 - 10 10 10 - 16(3) ω-4 20 20 10 tr. - 20 20 20 - 16(4) ω-1 10 tr. - 10 20 20 17(1) 10 18(0) 30 10 10 10 10 20 10 10 10 10 18(1) ω-9 40 20 30 10 20 30 20 10 50 10 18(2) ω-6 40 30 20 20 10 20 10 10 40 20 18(3) ω-6 30 20 tr. 10 tr. 10 10 10 30 - 18(3) ω-3 40 10 20 30 10 10 40 40 18(4) ω-3 30 10 20 50 10 - 20 10 20 - 20(0) 10 tr. 10 10 20(1) ω-9 10 10 20 20 20(2) ω-6 tr tr. - 10 10 10 20(3) ω-6 10 10 10 10 10 10 20(4) ω-6 10 10 10 20 40 10 10 10 20(4) ω-3 10 10 10 10 10 20(5) ω-3 30 30 20 30 20 20 30 10 22(0) 10 22(1) ω-9 10 10 10 10 22(5) ω-6 10 22(5) ω-3 20 10 10 10 tr 10 22(6) ω-3 10 10 30 Table 3.12 Maximum percentages of various fatty acids sorted by algal class, numbers in brackets are for exceptions, dashes mean not present, spaces mean not analysed, adapted from Borowitzka et al.242

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3.3.2 THE EFFECT OF NUTRIENT AND ENVIRONMENTAL LIMITATIONS

ON LIPID LEVEL AND FATTY ACID PROFILE

Many microalgae growing under nutrient deprivation show enhanced lipid contents. Out of the nutrients nitrogen has the most influence but other nutrients can also be limited to achieve an enhanced lipid content, for the Diatoms, silicon limitation will increase the lipid yield. It should be noted that limitation can also decrease the total lipid yield. In Dunaliella sp. or Turaselmis suecica a decrease in the nitrogen increases the carbohydrate levels at the expense of the lipids.241 It should be noted that a limitation in certain nutrients can limit the growth of the cells and therefore give a lower total lipid content overall. Generally speaking the green algae and diatoms respond well to the low nitrogen conditions though the level of lipid gain is highly variable across species (Table 3.13).

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Lipid Content (% dry wt) Algal Type Species +N -N Cyanophyceae Spirulina platensis 21.8 11.2 (Cyanobacteria) Anacystis nidulans 14.8 14.3 Chlorophyceae Ankistrodesmus sp. 18.3 40.3 (Green Algae) Botryococcus braunii 44.5 54.2 Chlamydomonas applanata 18.2 32.8 Chlorella pyrenoidosa 13.4 29.2 “ 10.0 70.0 “ 14.4 35.8 “ 20.0 86.0

Chlorella vulgaris (NH4) 11.8 52.8

“ (NO3) 21.8 57.9 Chlorella luteoviridas 17.5 28.8 Chlorella capsulata 11.7 11.4 Dunaliella primolecta 23.1 16.6 Dunaliella salina (UTEX 200) 25.3 9.2 Nannochloris sp. 20.8 35.5 Oocystis polymorpha 20.2 47.8 Ourococcus sp. 12.6 34.7

Scenedesmus obliquus (NH4) 27.0 49.5 Tetraselmis suecica 22.4 34.6 Hymenomonas carterae 20.0 14.3 Bacillariophyceae Cyclotella cryptica 23.0 36.8 (Diatoms) Nitzchia palea 22.2 39.5 Phaeodactylum tricornutum 20.0 24.0 Skeletonema costatum 23.8 30.3 Thalassiosira weissflogii 22.2 24.0 Chrysophyceae Iscochrysis sp. (UTEX 2307) 7.1 26.0 (Golden algae) Isochrysis galbana 23.0 23.1 Monallanthus salina 40.8 72.2 Cryptophyceae Cryptomonas rufescens 12.2 16.8 Eustigmatophyceae Monodus subterraneus 20.0 40.0 Rhodophyceae Porphyridium cruentum 98 (µL cell-1) 176 (µL cell-1) Table 3.13 Effect of nitrogen limitation on lipid content for a range of algal species, adapted from Borowitzka et al.242

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3.3.2.1 Cyanobacteria

A range of filamentous cyanobacteria (Anabaena sp. ATCC 33047, Anabaena variabilis, Anabaenopsis sp., Nodularia sp. (Chucula), Nostoc commune, Nostoc paludosum, Nostoc sp. (Albufera). Nostoc sp. (Caquena), Nostoc sp. (Chile), Nostoc sp. (Chucula), Nostoc sp. (Llaita), Nostoc sp. (Loa)) were studied for there lipid and fatty acid content. The lipid content never was higher than 13% and was at its highest under nitrogen depletion in the transition phase. The fatty acid profile of these lipids are given below in Table 3.14.

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Fatty Acid Profile (% of lipids) Strain 14(0) 16(0) 16(1) 16(2) 16(4) 18(0) 18(1) 18(1) 18(2) 18(3) 18(3) 18(3) 18(4) 18(4) 20(5) ω-4 ω-1 ω-7 ω-9 ω-6 ω-3 ω-4 ω-6 ω-1 ω-3 ω-3 Anabaena sp. ATCC 33047 0.4 42.3 7.8 0.3 3.7 0.9 0.9 2.6 5.7 35.3 Anabaena variabilis 0.4 37.4 15.8 0.2 0.9 4.3 8.6 32.3 Anabaenopsis sp 0.3 45.5 3.7 0.3 0.9 8.2 16.2 16.8 3.6 4.6 Nodularia sp(Chucula) 0.3 42.8 9.5 0.4 1.1 0.6 4.2 22.8 18.5 Nostoc commune 0.3 39.5 10.3 0.4 0.4 1.4 1.1 5.2 17.6 24.0 Nostoc paludosum 0.4 35.4 18.0 0.2 0.2 1.0 1.3 11.9 19.1 12.4 Nostoc sp. (Albufera) 0.3 50.8 3.3 0.3 0.1 0.7 0.2 2.6 6.5 11.6 3.2 19.0 1.4 Nostoc sp. (Caquena) 0.4 34.2 20.9 0.3 1.4 1.7 17.9 23.2 Nostoc sp. (Chile) 0.3 43.1 8.8 0.2 0.9 0.8 9.6 15.7 0.5 18.0 0.3 1.9 Nostoc sp. (Chucula) 0.3 30.9 19.5 0.3 0.3 1.0 2.5 10.0 19.3 15.8 Nostoc sp. (Llaita) 0.5 30.7 20.2 0.4 0.2 1.2 1.9 9.5 15.9 19.5 Nostoc sp. (Loa) 0.4 30.1 21.9 0.4 1.3 1.0 1.6 11.5 15.5 16.4

Table 3.14 Fatty acid profile of a range of nitrogen fixing cyanobacteria under normal conditions taken from Vargas et al.248

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3.3.2.2 Green Algae

Botryococcus braunii Though this algae generally produces hydrocarbons it can also produce fatty acids in small amounts (~ 10% dry weight). The highest yields of biomass and lipid concentration are achieved using KNO3 as the nitrogen source. The majority of these fatty acids were 16(0) and 18(1) ω-9 yet interestingly traces of methyl substituted fatty acids were also found.249

Parietochloris incisa This species has long been known as a source of arachidonic acid (20(4) ω-6). Under extreme nitrogen starvation the fatty acid content of the cells reached 35 % dry weight with AA making up at least 60% of this number.250

Light Intensity Nitrogen Conditions Factor (µmol photons m-2 s-1) +N -N Biomass 35 4.3 4.5 (mg / ml) 200 6.3 5.8 400 7.9 4.2 Total Fatty Acids 35 17.5 25 (% dry weight) 200 22.5 30 400 35 22 AA content 35 7.5 15 (% dry weight) 200 11 17 400 13 14 Table 3.15 FFA content of Parietochloris incisa as a function of both light intensity and nitrogen limitation

Parietochloris incisa produces more lipids under nitrogen deficient conditions except when a high intensity light is used. Under the optimum conditions for high lipid content arachidonic acid (C20(4)) is produced in the highest quantities.251

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Chlorella vulgaris Many studies have been carried out on Chlorella vulgaris and its lipid content. Nitrogen depletion does increase lipid yields while the major lipids tend to be C16(0), C18(0), C18(1) and C18(3) ω-6.252, 253 Iron limitation is also a significant factor in the growth of Chlorella and the lipid accumulation. Liu et al. found that by using between -5 -8 -1 1.2 × 10 and 1.2 × 10 mol L of FeCl3/EDTA increased the total amount of cells by 25%. This additional iron also affected the lipid content with 1.2 × 10-5 mol L-1 of iron according a lipid content of 56.6 % (dry weight) compared with just 7.8 % when no iron was added.254

Ulva pertusa Both nitrogen and phosphate starvation effect the total amount of lipids in this algae. Though nitrogen starvation resulted in a loss of biomass and even negative specific growth rates by the 4th day both factors increased crude lipid content (% dry weight). It also increased the amount of monosaturated and unstaturated fatty acids. Phosphorous limitation did not effect lipid content.255

Dunaliella Salina NaCl inhibits growth of the D. salina cells when it is present at over 1.5 M yet increasing the amount of salt from 0.5 M (sea water) to 1.0 M increased lipid concentration to 70% from 60%. The majority of these lipids were triglcyerides.256

Nannochloris sp. Generally Nannochloris does show a percentage increase in lipids when nitrogen is depleted, however, these gains are by far outweighed in the drop in cell density observed (Table 3.16)

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Initial Nitrate Conc. Cells Lipids (%) TG (%) (mM) (g/L) 9.9 2.86 31 26 8.0 2.18 32 30 6.0 1.73 30 29 4.0 1.44 30 40 2.0 0.79 30 37 0.9 0.24 40 54 Table 3.16 The effect of nitrate on the cell growth and lipids in the stationary phase for Nannochloris spices, taken from Tagaki et al.257

Yamaberi et al. also studied Nannochloris sp. UTEX LB 1999 for the fatty acid content. They found that the maximum growth rate was under reduced KNO3 conditions however the triglyceride content was not affected significantly by nitrogen depletion except in the stationary phase where the TG content was 2.2 times higher than that in the nitrogen rich environment, This was not a large enough increase to justify the use of this algal strain for bio-oil production.258

2.3.2.3 Dinoflagellates

Crythecodinium cohnii The growth of this marine algae can be manipulated by the use of glucose or salt. The growth rate is unsurprisingly sensitive to NaCl levels and is highest at high salt concentrations, the fatty acid profile is highly variable (Table 3.17).259, 260

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NaCl 0 g L-1 NaCl 2 g L-1 NaCl 5 g L-1 NaCl 9 g L-1 NaCl 15 g L-1 NaCl 23 g L-1 Fatty acid 1† 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 12 (0) 3.6 14.6 2.8 1.8 5.1 1.2 1.6 0.9 1.7 1.5 1.3 2.1 2.1 0.5 2.5 2.6 0.6 2.2 14(0) 15.5 11.8 21.8 12.8 15.4 12.5 13.0 13.5 12.4 12.3 16.7 14.0 15.4 13.7 13.5 14.4 17.3 13.7 16(0) 30.6 27.1 25.2 20.8 31.5 20.1 22.1 20.3 20.7 20.3 19.1 21.5 19.4 22.4 20.7 19.8 19.5 21.1 18(0) 17.1 15.1 11.7 9.7 16.2 15.4 7.1 10.4 9.1 7.8 9.6 8.7 8.1 10.1 9.2 9.9 9.9 10.6 18(1) 2.4 1.3 1.6 1.2 0.8 0.2 0.7 0.1 0.1 0.9 0.1 0.1 1.7 0.2 0.1 0.4 0.1 0.0 22(5) 1.3 3.4 1.6 0.2 1.9 0.3 tr 0.2 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.1 0.3 0.2 22(6) 29.4 26.6 35.4 53.5 29.2 50.5 55.4 54.6 55.7 56.9 52.8 53.8 52.9 25.8 53.7 52.9 52.3 52.1 Total Fatty 9.2 4.5 3.6 6.3 10.3 9.2 7.4 6.8 8.9 10.1 8.3 9.5 8.6 6.0 9.0 8.6 6.7 7.4 Acid (% DW cell) Table 3.17 Effect of salt on the fatty acid profile of three strains of Crythecodinium cohnii , fatty acids are given as % of total fatty acid content259

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3.3.2.4 Diatoms

Cyclotella cryptica A reduction in nitrogen and silicon does reduce the growth rate of this diatom, however there is a large increase in lipid production when both of these nutrients are withheld. The fatty acid profile also changes and produces more small chain saturated fatty acids (14(0), 16(0) etc.).261

Chaetoceros gracilis This marine diatom is sensitive to both phosphorous and silicon deprivation. Though no data was found on the exact fatty acid profile of the algae, a large change was observed in the amount of lipids produced under starvation conditions. The healthy cells produced 2.79 pg cell-1 of lipid of which only 2.8% of them were triglycerides, the phosphorous starved cells had a lipid content of 3.9 pg cell-1 of which 52% were triglycerides, and even though the silicon deprived cells produced 5.73 pg cell-1 lipids only a trace of triglycerides were observed.262

Phaeodactylum tricornutum In this diatom the residence time in the reactor as well as nitrogen limitation affects the concentration of lipids, triglycerides and even the fatty acid profile significantly. Again this is a complicated example as reducing the nitrogen intake increases the yield of fatty acids at one residence time, yet decreases it for another (Table 3.18).263

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Dilution Rate (h-1)

(Residence time)

Fatty acid 0.006 0.012 0.024 0.036 0.012 0.012 0.024 0.024 (%) (7 day) (3.5 d) (1.75 d) (1.15 d) -0.5N -0.2N -0.5N -0.2N

Total TG 68 69 42 57 71 75 25 21 14(0) 8.4 7.8 8.4 7.6 8.1 8.7 10.7 11.3 16(0) 9.4 10.5 11.3 12.9 12.0 27.3 15.8 19.5 16(1) 25.8 22.7 20.4 17.9 22.5 30.1 24.6 28.1 16(2) ω-4 3.8 4.8 5.6 5.5 3.5 1.1 3.8 2.9 16(3) ω-4 8.2 9.9 10.5 10.8 9.0 2.3 5.9 2.9 16(4) ω-1 0.3 0.2 0.5 0.9 0.2 0.1 0.6 0.5 18(1) ω-9 0.9 1.3 0.7 0.5 1.0 3.6 0.7 1.3 18(1) ω-7 0.5 0.6 0.6 0.6 0.6 0.6 0.5 0.4 18(2) ω-6 2.6 2.6 1.6 1.5 2.1 1.5 1.6 1.5 20(4) ω-6 0.9 0.4 1.1 1.1 0.5 0.1 0.3 0.1 20(5) ω-3 25.7 25.3 27.8 29.0 27.9 14.7 22.5 20.0 22(6) ω-3 1.8 1.7 2.0 2.5 2.0 1.0 2.2 1.9 short chain 6.3 6.8 5.9 5.3 5.0 1.3 3.9 2.2 <14(0) Other 5.4 5.5 3.9 4.0 5.6 7.7 7.0 7.6 Table 3.18 Effect of residence time and low nitrogen conditions on fatty acid profile of Phaeodactylum tricornutum adapted from Alonso et al.263

Phaeodactylum tricornutum is also capable of mixotrophic growth and the addition of glycerol has an effect on lipid concentration and the fatty acid profile (Table 3.19). The algae was grown in a 1 L bioreactor under a light intensity of 165 µmol m-2 s-1, the maximum yield of algae was 16 g L-1. Further studies on EPA production was undertaken by Yongmanitichai et al.264

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Fatty acid Glycerol Concentration (M) Ammonium Addition

+ (% dry weight) Con. 0.005 0.01 0.05 0.1 Con. NH4

Total fatty acid 7.24 7.83 8.94 9.61 16.98 15.52 22.17 14(0) 0.49 1.0 1.85 2.30 2.59 2.25 0.82 16(0) 1.10 0.71 1.13 1.62 3.98 3.65 4.63 16(1) 2.92 2.07 2.33 3.16 5.08 4.48 11.17 18(0) 0 0.38 0.03 0.04 0.11 0.08 0.20 18(1) ω-9 0.07 0.45 0.81 0.10 2.92 2.46 1.92 18(1) ω-7 0.14 0.04 0.05 0.05 0.00 0.24 0.99 20(5) ω-3 1.90 2.37 2.38 2.05 2.04 2.15 2.27 Table 3.19 Effect of glycerol and ammonium on the fatty acid profile of Phaeodactylum tricornutum, taken from Ceron Garcia et al.265

Thalassiosira psudonana In this marine diatom, light intensity and the time of harvesting most effect the lipid composition. Lipid concentrations were highest during the stationary phase (up to 40%), these lipids also contained the highest amount of triglyceride (harvesting in the logarithmic phase yielded high levels of polar lipids). Cells grown under intense light were higher in PUFA. Low light conditions gave mainly monounsaturated and saturated fatty acids.247, 266 Silicon starvation had little effect on the lipid content yet phosphorous starvation resulted in a severe drop in the number of cells.267

3.3.2.5 Golden algae

Isochrysis Galbana The main energy storage in this algae is lipids whatever the level of nitrogen in the batch culture. There are some changes in lipid content when the amount of nitrogen is varied yet this algae is more sensitive to the source of nitrogen as well as the phase that the algae is harvested in (Table 3.20).

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- - Nitrate (NO3 ) Nitrite (NO2 ) Urea [(NH2)2CO]

Fatty acid L ES LS L ES LS L ES LS

Total 12 15 18 13 18 15 10 20 15 (% DW) 14(0) 19.23 14.44 10.27 18.51 12.70 12.50 19.93 10.25 9.80 15(0) 0.26 0.27 0.32 0.21 0.22 0.33 0.29 0.24 0.34 16(0) 14.94 12.58 18.62 13.86 12.58 18.81 17.74 12.17 18.00 16(1) 20.50 19.43 24.59 20.55 19.09 25.84 19.92 12.43 21.58 16(2) ω-7 0.41 0.33 0.22 0.40 0.34 0.20 0.32 0.41 0.28 16(2) ω-4 0.41 0.16 0.14 0.42 0.17 0.14 0.45 0.11 0.09 16(3) ω-6 tr 0.16 0.10 - - 0.40 tr 0.11 0.14 18(0) 0.52 0.42 0.67 0.79 0.20 0.54 tr 0.15 0.52 16(3) ω-4 tr tr tr tr 0.13 tr tr 0.07 tr 18(1) ω-9 1.78 1.41 2.37 1.48 1.29 2.02 1.53 1.17 2.07 18(1) ω-7 1.76 1.68 3.88 1.99 2.11 4.08 1.79 1.17 2.66 18(2) ω-6 2.00 0.68 1.23 1.95 0.91 1.10 2.01 0.43 0.96 18(3) ω-6 0.54 0.33 0.41 0.96 0.26 1.18 0.61 0.20 0.35 20(0) + - 0.29 tr 0.22 - 0.73 0.37 - - 18(3) ω-4 18(3) ω-3 1.37 1.23 0.68 1.04 1.03 0.73 1.19 1.88 0.67 20(1) ω-9 tr tr 0.25 1.24 0.13 0.27 tr tr 0.15 18(4) ω-3 12.03 14.28 8.30 11.71 13.99 7.06 12.13 15.37 9.78 18(4) ω-1 0.51 0.59 0.38 0.43 0.16 0.38 tr - tr 20(2) ω-6 0.41 tr 0.39 tr 0.14 0.35 tr 0.34 0.15 20(3) ω-6 - - 0.34 - - 0.19 - - 0.45 20(4) ω-6 tr tr 0.31 1.04 0.28 0.33 tr 0.34 0.43 20(4) ω-3 tr tr - tr tr tr tr tr tr 20(5) ω-3 14.64 19.09 16.14 14.94 22.74 13.10 12.76 27.66 19.69 21(5) ω-3 2.23 1.49 0.51 0.65 0.98 1.15 1.65 0.95 0.64 22(5) ω-3 - 1.64 0.48 - - 0.51 - - 0.56 22(6) ω-3 5.64 7.85 8.26 6.30 10.14 5.85 5.14 14.13 9.94 Table 3.20 Fatty acid composition (% of total fatty acids) for I. galbana under differing conditions. L: logarithmic, ES, early stationary phase, LS, late stationary phase adapted from Fidalgo et al.268

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3.3.2.6 Eustigmatophyta

Ellipsoidion sp This marine algae has a high lipid content under nitrogen rich conditions, this is

coupled with a high growth rate. NH4Cl was shown to be the best nitrogen source giving a growth rate of 0.31 hr-1 and a total lipid concentration of 33.3% (compared to 8% for the control with no nitrogen source.) The fatty acid profile is given below.

Pre-logarithmic phase Post-logarithmic phase

N - source 0.32 0.64 1.28 1.92 2.56 0.32 0.64 1.28 1.92 2.56 (mmol L-1)

14(0) 3.70 4.39 4.54 4.49 5.40 3.67 3.57 4.31 8.91 5.71 14(1) 0.36 0.56 1.38 1.47 1.03 0.09 0.94 0.70 16(0) 37.9 25.5 15.7 16.5 13.7 42.3 40.8 26.5 18.4 18.6 16(1) 30.3 28.25 21.1 20.8 22.6 30.6 31.3 24.2 23.9 25.0 18(0) 0.84 0.54 0.57 0.94 0.89 1.15 0.57 18(1) ω-9 4.20 2.58 1.66 1.54 1.23 7.74 6.71 3.53 2.27 2.17 18(1) ω-7 0.92 1.12 1.45 1.40 1.27 0.93 0.85 1.17 1.44 1.45 18(2) ω-6 0.65 1.16 1.98 1.29 1.62 0.82 0.61 1.00 1.29 1.03 18(3) ω-3 0.47 1.49 2.77 3.11 4.27 0.07 20(4) ω-6 2.99 3.01 3.23 3.71 3.26 1.71 2.23 4.24 3.84 3.24 20(5) ω-3 9.51 19.9 34.1 39.0 38.9 5.44 7.17 18.5 28.4 29.6

Table 3.21 Fatty acid profile for the marine algae Ellipsoidion sp under NH4Cl rich conditions taken from Xu et al.269

3.3.3 HYDROCARBON CONTENT

The hydrocarbon concentration in algae is generally less than 5% (dry weight), however the green algae Botryococcus braunii produces a range of hydrocarbons, with levels of up to 90% dry weight having been observed.242 The majority of these hydrocarbons are extracellular, included in the colony matrix and occluded into globules. All aspects of Botryococcus braunii has been extensively reviewed by Banerjee et al among others.249, 270 There are three specific ‘races’ of Botryococcus braunii each characterised by their hydrocarbon content (Fig. 3.4-3.6).

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Fig. 3.4 Range of hydrocarbons produced by Race A Botryococcus braunii, taken from Banerjee et al.270

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Fig. 3.5 Hydrocarbons found in Race B B. braunii, taken from Banerjee et al.270

Fig. 3.6 Hydrocarbon found in Race L B. braunii, taken from Banerjee et. al.270

3.3.4 WAX ESTERS

Though found in some higher plants wax esters are not very prevalent in algae, two species have been shown to have the capacity to synthesise small amounts these are Chroomonas salina and Euglena gracilias.242

3.3.5 STEROLS

Sterols are highly important commercial products. They are generally used in the synthesis of steroid hormones and related compounds. Sterols from plants are sometimes referred to as phytosterols, as opposed to zoosterols from animals. A large number of these can be derived from algae (Table 3.22). This is not a complete list and many algae contain more than one including many which are not listed below. The typical concentration of sterols is in the range 1-4% (dry weight). Stanols also occur but are much rarer.242

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Trivial Name Chemical Name Algae Class Brassicasterol 24-methyl cholest-5,22-dien-3β-ol Diatoms Cyanobacteria Cholesterol Cholest-5-en-ol Red algae Chondrillasterol Stigmasta-7,22-dien-3-ol Green algae Clinasterol 24S-ethylcholest-5-en-3β-ol Cyanobacteria Dinosterol 4α,23, 24-trimethyl-5α-cholest-22-en-3β-ol Dinoflagellates Green algae Ergosterol 24R-methylcholesta-5,7,22-trien-3β-ol Red algae Golden algae Poriferasterol 24R-ethylcholesta-2,22-dien-3β-ol Diatoms Sitosterol 24R-ethylcholesta-5-en-3β-ol Yellow-green Stigmasterol 24S-ethylcholesta-5,22-dien-3β-ol Green algae Table 3.22 Main sterols found in algae, arrange by class, adapted from Borowitzka et al.242

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3.4 POLYMERS

3.4.1 POLYOLS & CARBOHYDRATES

Microalgae produce a vast array of carbohydrates which can be divided into two groups, the first are low molecular weight polyols containing glycerol or mannitol which act as osmotic effectors and large storage or structural polysaccharide polymers such as cellulose or starch.

3.4.1.1 Osmotically Active (Low Molecular Weight)

All algae will produce small low molecular weight carbohydrates in the growth cycle, with some species of Dunaliella being recorded as producing 50% by dry weight (Table 3.23). As these polyols are generally used by the organism for osmotic regulation, high salinity (and therefore any species which is impervious to high salt conditions) yield the highest levels. These polyols are an excellent food source for heterotropic growth so harvesting the full potential yield of polyol produced can be difficult.

ne rol rol ai e t et l a l -t at ctose) b -glycerol u ose - m ito ose ose ito e a n t ane al

Algae b (fr nn r uc ut m o a ctosyl S Gl osyl-glyce Glycerol Man Ma ohex Treh a ut rose L-Gl ycl Gluc Suc C L-Gl a-Gal

Cyanophyceae Freshwater X X X Marine X X X X Chlorophyceae X X X X Prasinophyceae X X Eustigmatophyceae X Baccillariophyceae X Chrysophyceae X X Table 3.23 Major polyol components accumulated by microalgae taken from Borowitzka et al.150

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3.4.1.2 Polysaccharides

These polymers are complex carbohydrates, made up of monosacchardide units bound together by glycosidic bonds (ether linkages). Polysaccharides can be homo- (all monomer linkages are the same) or hetero- (the monosaccharide varies in the chain).

The general formula of a polysaccharide is Cn(H2O)n-1, examples of polysaccharides are starch, glycogen, cellulose and chitin (structural examples are given in Appendix I). Polysaccharides are mainly uses as viscosifers, lubricating agents and flocculating agents. All algae produce polysaccharides, but for commercial production algal cells which excrete it into the environment are thought to be the most promising. There is extensive literature detailing the composition of polysaccharide in microalgae yet little detail on this as a commercial application. The important classes of algae which produce extracellular polysaccharides are Porphyridium (specifically P. aerugineum and P. cruentum), Chlamydomonas, Palmella, Katodinium, Phaeocytis, Anabaena flos-aquae among others. Members of these classes have been shown to excrete up to 90% of the polysaccharide into their environment, this equates to 10 g m-2 d-1 or 25 tonnes ha-1 a-1, typical molecular weights can range from 1800-5 x 106.150

Cyanobacteria as a whole produce complex exo-polysaccharides containing monosaccharide’s as well as various other pentose’s (sugars with five carbon atoms) these polysaccharides are also predominantly acidic containing acidic sugars, organic and inorganic acid groups. Research has also been focussed on applications of these polysaccharides in biomedical and metal extraction. It should also be noted that for most algae strains higher polysaccharide production is achieved under low nitrogen conditions.271, 272 Larger polymers (2 x 106) have been reported including proteins, fucoidan polysaccaharides and fatty acids. A potential application of this area as bioflocculants suitable for safe water cleaning These bioflocculants generally occur in the cyanobacteria.273

3.4.2 BIOPOLYMERS FROM ALGAE

Some species of cyanobacteria have the ability to synthesise a range of polyhydroxyalkanoates (PHA).274 PHA from cyanobacteria can be made up from over

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150 monomers three of the most common structures, polyhydroxy butyrates (PHB), are given below in Fig. 3.7.

R O

* * x O n

General PHA formula

O O O O

* * * O x O O * x y * * x y O P(3HB) P(3HB-co-3HV) P(3HB-co-4HV)

Fig. 3.7 Common biopolymers found in cyanobacteria

PHA content in naturally occurring cyanobacteria is around 6wt% however, under optimum conditions this can be increased to around 10 wt%. Genetically modified organisms can produce even more and levels of up to 60 wt% have been reported. The main PHA producing species include Chloroglorea fritschii. Spirulina platensis, Aphanocapsa sp. Anacystis cyanea, Synechococcus sp. Synechocystis sp. and Nostoc sp.275

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3.5 GENERAL HUMAN AND ANIMAL NUTRITION

Algae have been used as a source of protein and carbohydrates by humans and animals for over 2000 years. The unpredictable nature of algal blooms and difficulties with consistently farming algae meant that this natural abundant source of protein was soon replaced with more reliable sources. Nowadays algal products are sold generally as high value nutraceuticals in the form of tablets, capsules or suspensions. The three main species of algae grown for this purpose are Spirulina, Chlorella and Dunaliella.276 The high protein content of algae (as well as the natural products described previously) is well known and is a significant factor in considering algae as a source of nutrition. The amount of protein can be difficult to measure and so figures in the literature have to be treated with caution, however comparisons across food groups are possible (Table 3.24).277

Commodity Protein Carbohydrate Lipid Yeast 39 38 1 Meat 43 1 34 Milk 26 38 28 Rice 8 77 2 Soybean 37 30 20 Anabaena cylindrica 43-56 25-30 4-7 Chlamydomonas 48 17 21 rheinhardii Chlorella vulgaris 51-58 12-17 14-22 Dunaliella salina 57 32 6 Porphyridium cruentum 28-39 40-57 9-14 Scenedesmus obliquus 50-56 10-17 12-14 Spirulina maxima 60-71 13-16 6-7 Synechococcus sp. 63 15 11 Table 3.24 General composition of food sources and selected algae (dry weight %) taken from Becker277

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There are also certain toxicological aspects which must be considered when a material is being considered as a novel food source. Biogenic toxins include DNA and RNA and in the US the recommended limit of total nucleic acids from all sources must not exceed 4.0 g per day. This would be equivalent (in supplement to a normal diet) to 20 g of algal matter per day. Algae also are highly efficient at binding heavy metals (Section 3.7) and this can cause a health risk in algae which has been farmed from heavily polluted areas. This would not effect algae grown in bioreactors or other controlled environments. Certain algae also produce other harmful toxins, these generally are cyanobacteria, none of these algal toxins have been reported in any strain which is under current mass production.241 One of the final problems with using algae as a food source is the digestibility of the cell wall. As cellulose is one of the main components of this wall, and cannot be broken down by humans or other non-ruminants, most algae need some sort of processing to prepare them sufficiently.278 A number of trials have taken place on using algae as feed for poultry, pigs and rudiment animals. Generally no serious problems with using algae were reported but a drop in the uptake of carbohydrates was observed for most animals tested.241, 279-282

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3.6 FUELS

Due to the increasing pressure for fuel security, to reduce global emissions of greenhouse gases and of the rising cost of crude oil, alternative transport and heating fuels are being sort. It is possible to use the dried biomass matter as a fuel by direct combustion in its own right. Demirbas et al. estimated that over 97% of the worlds bio-energy production comes from this direct method.283 Investigations have since shown that this method is uneconomical and that upgrading into a more efficient fuel is more practical.284 The first generation biofuels were derived from higher plant biomass, however fears over deforestation, competition with food sources and negligible carbon savings has spurned new interest into alternative feedstocks such as microalgae.285, 286 A list of the possible fuels derived from microalgae is given in Table 3.25.

Fuel Type Source Electricity Biochemical fuel cells Biodiesel Liquid for transportation Triglyceride Bioalcohols Liquid for transportation General biomass by fermentation Hydrocarbons Liquid for transportation Formed in certain species (need to be cracked) Bio-oil Liquid for Heating Pyrolysis/liquefaction of biomass Direct Use Emulsified liquid fuel general biomass Power generation Methane Gas Anaerobic digestion Hydrogen Gas Anaerobic digestion Formed by certain species Table 3.25 Possible fuels derived from algae

3.6.1 DIRECT USE

Direct combustion of biomass is a process still widely used around the world, and 35% of all energy in developing countries comes from this method.287 Dried biomass from algae can also be used in this way, to produce heat or when supplied at a

99 Chapter 3: Products and Technology constant rate to produce steam for electricity generation. This, however, is inefficient and labour intensive.288 Diesel engines have been shown to run efficiently while using pulverised coal, with a particle size of no more than 8 µm.289 More recently a biomass slurry (particle size between 30-70 µm) has been proposed as an alternative diesel fuel.290 However, unicellular algae have cell sizes which range from 3-10 µm, this is ideal for combustion in a diesel engine. Scragg et al. found that the direct suspension of dried algae was not suitable for use and that the algae (Chlorella vulgaris) was concentrated into a thick slurry before making an emulsion with the fuel. A surfactant was also added to stabilise the solution. The resulting emulsion was much less viscous than diesel fuel (or vegetable oil) and as such was deemed unsuitable for use in a modern common rail injection engine.291 Other emulsification agents can be used, such as the naturally occurring enzyme NOE-7F, which has been shown to efficiently create water/bio-solution/diesel fuel emulsions.292, 293

3.6.2 BIO-OIL

One efficient method of converting biomass to liquid hydrocarbons is by (anhydrous) pyrolysis. This process can be divided into three subsections

1. Conventional Pyrolysis (carbonization) 2. Fast Pyrolysis 3. Flash Pyrolysis

The first process uses slower reaction times and higher temperatures and results in a mixture of solids, liquid and gas. The second and third process use temperatures between 350-600 °C and residence times normally less than two seconds. These processes have much higher liquid fractions. The mechanism of pyrolysis is complicated and biomass derived from different sources are converted by different mechanisms (cellulose, lignin etc.) it is out of the scope of this review to discuss them, however, several thorough reviews have been published in this area.294 The pyrolysis of green algae species Chlorella protothecoides and Cladophora fracta has been investigated. Temperature was found to be highly important on the final yield of liquid fuel for both species, 500 °C gave the highest yields of just over

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50%.295 It should be noted that higher yields than this have been reported from bio- oils derived from Spirulina when an iron sulphate catalyst was used.296 A more in depth study was carried out on Chlorella protothecoides grown both autotrophically (AC) and heterotrophically (HC) with glucose.297, 298 The protein, lipid and carbohydrate contents prior to pyrolysis were markedly different (Table 3.26).

Component AC HC Protein 53 10 Lipid 15 55 Carbohydrate 11 15 Table 3.26 composition of Chlorella protothecoides prior to pyrolysis

This difference in composition also yielded a very different bio-oil composition. The HC grown algae not only gave a larger oil fraction (58%) but also an oil which has much more similarity to crude oil then the bio-oil produced from conventional biomass feedstocks (Table 3.27).

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Bio oils Fossil Oil Properties Wood AC HC C 56.4 % 62.07 % 76.32 % 83.0-87.0 % H 6.2 % 8.76 % 11.61 % 10.0-14.0 % O 37.3 % 19.43 % 11.24 % 0.05-1.5 % N 0.1 % 9.74 % 0.93 % 0.01-0.7 % S n.d. n.d. n.d. 0.05-5.0% Density (kg l-1) 1.2 1.06 0.92 0.75 – 1.0 Viscosity (Pa s, 40 °C) 0.04-0.20 0.10 0.02 2-1000 Heating Value (Mj Kg-1) 21 30 41 42 Stability Low Medium Medium High Chemical Composition Sat. Hydrocarbons 0.86 % 1.02 % Aromatics 0.75 % 2.21 % Polars 33.82 % 89.93 % Asphaltenes 64.57 % 6.94 % Table 3.27 Elemental, chemical and physical properties of a range of bio-oils from fast pyrolysis of biomass, adapted from Miao et al.297

The heterotrophic growth of certain algae has been shown to increase the lipid fraction as well as increasing the biomass available. The research presented above suggests that a higher lipid content yields a higher liquid bio-oil fraction as well as creating a higher quality fuel oil.

3.6.3 BIODIESEL

Generally the term biodiesel is used to refer to the fatty acid alkyl esters (FAAEs) derived from the glyceride portion of the lipid layer by transesterification. The triglyceride molecule is the main constituent of vegetable and algal oils, and can be transesterified with an alcohol over a basic or acidic catalyst to give FAAEs. Vegetable oil is almost entirely made up of triglycerides where algal oil can contain phospholipids and other related compounds. Methanol is most commonly used to make biodiesel as it is less expensive than other alcohols. Though theoretically three

102 Chapter 3: Products and Technology molecules of alcohol are needed for every molecules of triglyceride, usually an excess of up to twelve molecules is used. The chain length and level of saturation of the FAAEs produced depends solely on the feedstock used. Biodiesel has been extensively reviewed and every aspect involved in the synthesis to the combustion of this fuel discussed.299, 300 Due to the high efficiency of converting solar energy to lipids under optimised conditions algae could theoretically produce up to 30 times more oil per hectare than the terrestrial oilseed crops (Table 3.28). This high productivity coupled with the ability to grow on land unsuitable for alternative cultivation make algae a promising alternative for biodiesel production.301-303 Potentially heterotrophic growth can be utilised to increase production of either the total lipid content or the more desirable fatty acids present in the oil. Though this appears less efficient than using sunlight alone, it is also a potential use for glycerol produced from the process.

Crop Oil Yield (L ha-1) Land Area Existing US Necessary (M ha) Cropping Area (%) Corn 172 1540 846 Soybean 446 594 326 Canola 1190 223 122 Jatropha 1892 140 77 Coconut 2689 99 54 Oil Palm 5950 45 24 Microalgae 136900 2 1.1 (70 wt% oil) Microalgae 58700 4.5 2.5 (30 wt% oil) Table 3.28 Comparison of biodiesel sources, taken from Chisti et al.302

The amount of glycerides obtainable differs depending on species, nutrients and growth conditions, these factors were discussed in more detail in Section 3.3. Biodiesel derived from algae has the potential to contain more polyunsaturated esters than that derived from vegetable oils, though this will lower the oxidative stability the vitamin E present in algae oils (or additional antioxidant) would potentially

103 Chapter 3: Products and Technology compensate. Legally the European legislation EN14214 limits the amount of linolenic acid ester to 12 mol% where there are also limits placed on the iodine value. European legislation also requires that FAME with 4 or more double bonds are limited to 1 mol%. This legislation does not apply when using biodiesel as heating oil. Partial hydrogenation of the FAME chain is a plausible technology, though additional production costs would be occurred. It is also likely that with the development of algal biodiesel and a better understanding of the physical properties of the fuel that this legislation will be changed.302 One potential difference in algal biodiesel is an improved low temperature behaviour as PUFA tend to have very low freezing points. Xu et al. grew samples of Chlorella protothecoides under differing conditions. the algae grown with glucose achieved a lipid concentration of 55%. The resulting lipids were then converted into biodiesel and the fatty acid profile (Table 3.29) and other physical properties (Table 3.30) were investigated.304

FAME % Abundance Myristic acid methyl ester C14(0) 1.31 Palmitic acid methyl ester C16(0) 12.94 Heptadecanoic acid methyl ester C17(0) 0.89 Linoleic acid methyl ester C18(2) (ω-6) 17.28 Oleic acid methyl ester C18(1) (ω-9) 60.84 Stearic acid methyl ester C18(0) 2.76 10-Nonadecenoic acid methyl ester C19(1) (ω-9) 0.36 11-Eicosenoic acid C20(1) (ω-9) 0.42 Eicosanoic acid methyl ester C20(0) 0.35 Table 3.29 Fatty acid profile of the biodiesel produced by Chlorella protothecoides published by Xu et al.304

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Properties Algal Oil EN 590 EN 14214 ASTM D6751 (Diesel fuel) Density (g cm-3) 0.864 0.820-0.845 0.860-0.900 K. viscosity mm2s-1 (40 °C) 5.2 2.0-4.5 3.5-5.0 1.9-6.0 Flash Point (°C) 115 55 min 120 130 Pour Point (°C) -12 -10 CFPP (°C) -11 -5 Acid value (mg KOH g-1) 0.374 0.5 (max) 0.5 max 0.8 max Heating Value (MJ kg-1) 41 40-45 Table 3.30 Comparison of physical properties of the algae biodiesel produced by Xu et al.304

As discussed in more detail in Section 3.3 the fatty acid profile of any particular algal oil is highly dependent on both the strain and the conditions it has been grown under. Certain fatty acid methyl esters are more suitable as biodiesel than others. Knothe reasoned that oleic acid methyl ester and palmitoleic acid methyl ester (C16(1) and C18(1)) are the most suitable FAME for biodiesel.305 Though it is difficult to estimate the physical properties of any sample of biodiesel, especially given the paucity of information on the long chain esters, it seems likely that while increasing the lipid yield of the algae is important, an algal oil high in monounsaturated esters, low in saturates with an increased amount of polyunsaturates (though still a large amount of vitamin E) would potentially yield the best biodiesel possible.

3.6.4 HYDROCARBON FORMATION

As previously discussed certain algae, namely Botryococcus braunii, are capable of producing hydrocarbons amounting to over 70% dry weight. These hydrocarbons are listed above in Section 3.3.3. These hydrocarbons are unsuitable as a liquid fuel and must be upgraded to petrol, diesel or bio-oil fuels by hydro- or catalytic cracking.306 For these processes to work, however, the cells must be harvested and dried. Alternatively liquefication of the wet cells can be achieved when they are heated to 300 °C, over a sodium carbonate catalyst, with additional water, this is then followed

105 Chapter 3: Products and Technology by a solvent extraction. This method yielded a 70 % (dry weight) yield of hexane soluble oil, with an elemental analysis of C: 84.1%, H: 14.3%, N: 0.9%, O: 0.7%. The heating value was 49 MJ Kg-1 (higher than diesel or petrol) and the viscosity was 64 mPa s-1 at 50 °C (akin to a thick waste vegetable oil).307 Tsukahara et al. investigated the energy balance of a liquid fuel made from Botryococcus braunii if the algae was grown using the waste heat and flue gases from a coal power plant as well as a waste water stream rich in nitrates and phosphates. Assuming the energy efficiency of the coal plant is 30%, the heating value of coal is

28 MJ/kg, the carbon content of the coal is 65 wt% and that 30% of the CO2 in the flue gas can be used by the algae as well as algal yield is 15 t dry weight/ ha/ year and that the density of dried algal matter is 0.5 kg / m3, the energy for liquification / energy of produced oil would by 0.15.

Fig. 3.6 Energy and carbon emissions for a coal power plant (A), and one mitigated by microalgal cultivation, taken from Tsukahara et al.308

3.6.5 BIOALCOHOLS

Bioethanol, produced by the fermentation of sugars or starch has long been used as a substitute for petrol fuel.309 Originally the microbial systems used could only convert specific sugar monomers directly. Though algae do produce sugar monomers, polysaccharides are more prevalent. As with biodiesel the use of first generation crops (corn, sugarbeet) put the alcohol fuel in direct competition with foodstuff. Due to this concern current research

106 Chapter 3: Products and Technology has focussed on producing pure ethanol from the far more abundant polysaccharide feedstock derived from non-food sources. There are four steps in the conversion of these natural polymers to pure alcohol.310

1. Hydrolysis of the polysaccharides to sugar monomers 2. Purification of sugar product (separating from lignin) 3. Fermentation to produce alcohol 4. Distillation to produce pure alcohol.

The first step can be achieved by using weak acids at high temperatures and pressures, strong acids at more benign temperatures and pressures or more recently, enzymatic hydrolysis which can be used at ambient temperatures and middling pH levels. The enzymatic conversion is the most efficient method and produces fewer by-products than the acid hydrolysis steps do. The fermentation step can be carried out by a range of organisms, though bakers yeast is the most common it can not produce ethanol from xylose, a standard sugar in most cellulosic materials. Currently a large amount of genetic engineering is being undertaken to address this problem.311 Further research in this field is also being directed towards organisms which are capable of producing cellulosomes, which convert cellulose and starches directly into ethanol.312, 313 A more promising alcohol than ethanol is biobutanol, which can be made from the same feedstocks, using a different organisms for the fermentation (Clostridium acetobutylicum, Escherichia coli). Biobutanol is less polar than ethanol has a higher energy content (88% that of petrol compared to 70% for ethanol), it can be used in higher blends without the need for engine modification and is less hygroscopic. The main drawback of biobutanol production is that an increased amount of butanol (up to 4%) kills the producing organisms.314 Though it is clear that alcohols from cellulose can contribute significantly to reducing greenhouse gas emissions and meeting current energy demands, the general cellulose materials are far more likely to come from grasses or other higher plants than algae.315, 316

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3.6.6 HYDROGEN

Certain algae (specifically the green and blue-green algae) have evolved the ability to extract protons and electrons from water, via the water splitting reaction of PSII (Fig.

3.8). In the green algae the protons and electrons are recombined to generate H2 gas by a chloroplast hydrogenase, this gas is then released from the algae broth in purities reaching 98%.317 Though it is out of the scope of this review to go into detail about hydrogen production a large number of reviews have been completed in this area. These include general hydrogen production from microorganisms including genetic manipulation, the effect of nutrients and the future developments necessary for large scale production.318-334 Certain microalga (Chlamydomonas reinhardtii) have the capability to also produce hydrogen peroxide. Hydrogen peroxide is an efficient and clean fuel used for rocket propulsion, motors and plausibly for heating. Hydrogen peroxide is produced by the photosystem in a catalyst cycle, with a redox mediator (methyl viologen), and oxygenated water.335, 336

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Fig. 3.8 Photosynthetic H+/e− flow in C.reinhardtii under aerobic (A) and anaerobic

(B) conditions in WT and H2 high-production mutant Stm6, taken from Hankamer et al.337

3.6.7 BIOGAS BY ANAEROBIC DIGESTION

Algal biomass can be converted into biogas by anerobic digestion and fermentation. The biomass can be continuously fed into a digestor, under anaerobic conditions, where mixtures of bacteria hydrolyse and break down the bio-polymers, other bacteria can then convert these into methane rich gas. The residence time for biomass in the reactor is around 200 days where different bacteria will break down the biomass. This process can be optimised by using bacteria which survive at two temperature ranges, mesophiles (~35 °C) and thermophiles (~55 °C). This process yields a mixture of methane, higher hydrocarbons, CO2 and H2S. With a steam reforming step this mixture can be converted to syngas (CO and H2) which must then be purified by

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membrane technology.338 Alternatively the methane can be used for electricity generation.339

3.6.8 GASIFICATION

If the biomass is heated to over 700 °C with air the primary products are CO and H2 gas. This ‘syngas’ can then be either used as a source of hydrogen, converted into methanol or conveted into alkanes via the Fisher-Tropsh process. 340 Biomass from dried Spirulina sp. was heated to 850, 900 and 1000 °C, and the gas composition analysed, the theoretical yield of methanol was then calculated (Table 3.31).341 The researchers concluded that gasification at 1000 °C yielded the highest gas concentration and therefore largest methanol fraction. However, 4/5 of the energy required to synthesise the methanol was taken up producing the algae and that significant improvements in the production process would yield a more favourable energy balance.

Gas Composition (% vol) Methanol

Temp. (°C) H2 CO CO2 CH4 C2H4 N2 O2 (g/g biomass) 850 34.5 18.0 32.9 10.7 2.1 1.6 0.2 0.50 900 35.9 14.5 36.4 9.7 0.7 2.6 0.3 0.46 1000 48.2 9.8 31.1 9.1 0.1 1.5 0.2 0.64 Table 3.31 Gasification and theoretical yield of methanol from Spirulina biomass adapted from Hirano et al.341

3.6.9 MICROBIAL FUEL CELL

A microbial fuel cell is a device that converts chemical energy to electrical energy by using the enzymes present in some microorganisms. At the anode a fuel (sugar, waste water, alcohol etc.) is oxidised by the organism generating electrons and protons. The electrons are then transferred via an electric circuit and the protons through a membrane to the cathode where oxygen is reduced to water.342 Cyanobacteria can be

used as the oxidizing organism, or more generally as a producer of H2 (see Section 3.6.6) to feed a conventional fuel cell.343-345

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3.7 MISCELLANEOUS USES OF ALGAE

3.7.1 BIOFOULING

As discussed previously, algae and particularly the cyanobacteria produce a variety of bioactive metabolites that have antibiotic, algicidal, antifungal and antimacrofouling properties. All of these functions have alleochemical functions in the natural environment, such as in the prevention of fouling by colonising organisms. Molecules with antifouling activity include fatty acids, lipopeptides, amides, alkaloids, terpenoids, lactones, pyrroles and steroids.159, 162

3.7.2 WASTEWATER TREATMENT

One way in which algae cultivation can be used to treat wastewater was developed by Oswald et al. in the 1950’s. If the waste water is passed through a series of ponds, with the necessary concentration of nutrients for algal growth, then the algae will produce oxygen allowing aerobic bacteria to break down the remaining contaminants in the water. This is highly efficient as it eliminates the need for mechanical agitation.346 The algae must be removed continuously for this technology to work which can be energy and cost consuming. Many of the methods discussed in Chapter 2 have been used historically however, immobilization of the algae cells seems the most promising technology.347, 348 Algae can also directly remove certain elements from the waste stream. Disease causing organisms, sediments, oxygen demanding wastes as well as organic material, nitrogen, phosphorous, sulphur and potassium from various industrial sources have the potential to be broken down by algae.349-352 Heavy metal contaminants in polluted water is also a large problem, traditionally these ions could be removed by binding to a ligand and precipitation, activated carbon adsorption. electrodialysis or reverse osmosis. Bioremoval of metals could be a significant replacement of these technologies. Algae can remove metals by one of two mechanisms. The first is Passive Uptake (metabolism independent), also known as bioadsorption. This occurs when the metal binds to the cell wall by either ion exchange or complex formation. Bioadsorption is dependent on ion concentration and the pH of the solution. The second mechanism is Active Uptake (metabolism

111 Chapter 3: Products and Technology dependent) this involves intercellular accumulation of the metal ions. These processes are energy dependent and sensitive to different parameters including pH, temperature, ionic strength, light, etc. They are inhibited by low temperature, absence of energy source, metabolic inhibitors, and uncouplers. This mechanism can work on concentrations less than 1 ppm.232 Both mechanisms are strain specific but a large number of metal ions including, copper, zinc, barium manganese, cobalt cadmium, nickel, strontium, mercury, silver, gold, lead and uranium have been shown to be removed in this manner.346, 350, 353, 354

3.7.3 CO2 SEQUESTRATION

The last feature of algae which make it an interesting technology is the ability to fix carbon dioxide (as well as other harmful greenhouse gases from flue gas). Microalgae is thought to be better at this than the terrestrial plants and estimates have placed the -1 -1 243, 319, 355 amount of CO2 fixable from between 0.2-3 g L day . However, the final use of this biomass and the processing involved will determine the actual CO2 sequested and only a full LCA will correctly determine the efficiency of cleaning flue gases in this manner.

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3.8 REFERENCES

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124 CHAPTER 4

GLOSSARY AND DEFINITIONS

AA Arachidonic acid, C20(4), ω-6 ACP The acyl carrier protein (ACP) is an important component in both fatty acid biosynthesis Allelic Forms of a gene, usually arising through mutation, that are responsible for hereditary variation. Allenic Compound with two C-C double bonds situated next to one another Autotrophic (Of organisms such as green plants) capable of manufacturing complex organic nutritive compounds from simple inorganic sources such as carbon dioxide, water and nitrates using energy from the sun Axenic (Of a biological culture or culture medium) free from other microorganisms uncontaminated Bacillariophyceae Diatoms Bioadsorption Uptake of compounds such as heavy metals by biological systems Carotenoids Organic pigments that are naturally occurring in chromoplasts of plants and other photosynthetic organisms. There are over 600 known carotenoids; they are generally split into two classes, xanthophylls and carotenes. Cellulosomes Enzymes capable of breaking down cellulose Chlorophyta Green Algae Chloroplast Organelles found in plant cells and eukaryotic algae that conduct photosynthesis. Cumulenic Compound with three C-C double bonds situated next to one another Cyanophyceae Cyanobacteria, blue-green algae

125 DG Diglycerides DGLA Dihomo-gamma-linolenic acid, C20(3) ω-6 DHA Docosahexaenoic acid – C22(6) DHA Docosahexaenoic acid, C22(6) ω-3 DPA Docosapentaenoic acid, C22(5), ω-3 EPA Eicosapentaenoic acid, C20(5) ω-3 ETA Eicosatetraenoic acid, C20(4), ω-3 ETE Eicosanotrienoic acid, C20(3), ω-3 Eukaryotic Organism having as its fundamental structural unit a cell type that contains specialized organelles in the cytoplasm, a membrane-bound nucleus enclosing genetic material organized into chromosomes, and an elaborate system of division by mitosis or meiosis, characteristic of all life forms except bacteria, blue- green algae, and other primitive microorganisms FFA Free Fatty Acids Filamentous Long slender chain of cells into which some algae are divided Flocculation A compound that causes aggregation of suspended particles / cells. FPR Flat Plate Reactor FTR Fermenter Type Reactors Heterotrophic (Of organisms such as animals) obtaining carbon for growth and energy from complex organic compounds HTR Horizontal Tubular Reactor Hydraulic Retention Time The length of time that a soluble compound spends (HRT) in the reactor LED Light emitting diode MG Monoglycerides Mixotrophic An organism capable of both Heterotrophic and Autotrophic behaviour Monosaccharide Simple sugar monomers

126 Motile Biological organism capable of moving spontaneously and independently Organelles A differentiated structure within a cell, such as a mitochondrion, vacuole, or chloroplast, that performs a specific function. PHA Polyhydroxy alkanoate PHB Polyhydroxy butanoate Photoautotropic Capable of using light as the energy source, in the synthesis of food from inorganic matter phycobiliproteins Light capturing proteins Polysaccharide Polymers formed by monosaccharide units Prokaryotic A cellular organism that has no nuclear membrane, and no organelles except ribosomes, its genetic material is in the form of single continuous strands forming coils or loops, Bacteria and blue-green algae. PUFA Polyunsaturated fatty acid Reynolds Number Re is a dimensionless number that gives a measure of the ratio of inertial forces to viscous forces and, consequently, it quantifies the relative importance of these two types of forces for given flow conditions. Stanol Fully saturated phytosterols Sterol a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring

TG Triglycerides VTR Vertical Tubular Reactor Xanthophylls Yellow pigments from the carotenoid group.

127 CHAPTER 5 – DIRECTORY

Name Area of Research Institution Email address A. Demirbas Transport fuels from algae Selcuk University, Department of Chemical [email protected] Engineering, TR-42031 Konya, Turkey A. Richmond Algal growth, including all aspects of Blaustein Institute for Desert Research biotechnology used for large scale Ben-Gurion University of the Negev, Sede Boker produciton Campus. Israel A.M.S. Mayer Marine Pharmacology Department of Pharmacology, Chicago College of [email protected] Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515, USA E. M. Grima Algal biotechnology, chemical University of Almeria, Department of Chemical engineering of algal bioreactors Engineering, Almeria 04071, Spain

E.W. Becker Biologist, algal protein Institut für Mikrostrukturtechnik, [email protected] Forschungszentrum Karlsruhe, Universität Karlsruhe, Hermann-von-Helmholtz-Platz 1, D- 76344 Eggenstein-Leopoldshafen, Germany. F.X. Malcata Reactor Design Escola Superior de Biotecnologia, Universidade [email protected] Cato´lica Portuguesa, Rua Dr. Anto´nio Bernardino de Almeida, P-4200-072

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Name Area of Research Institution Email address G. Knothe Biodiesel USDA National Centre of Agriculture Utilization [email protected] Research, Peoria, IL 61604 USA J.R. Bennemann Biohydrogen University of California Berkeley, Department of [email protected] Plant & Microbial Biology, Berkeley, CA 94720 USA M. A. Borowitzka Micro-algal biotechnology Environmental and Life Sciences, Murdoch [email protected] University, Perth, Australia M.G. Guerrero Reactor design to produce economically Instituto de Bioquímica Vegetal y Fotosíntesis, [email protected] important FFA Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Avda. Américo Vespucio 49, Sevilla, 41092, Spain N. Mallick Polymers from algae, immobilised algal Indian Institute of Technology, Agricultual and [email protected] cells Food Engineering Department, Kharagpur 721302, W Bengal, India U.C. Banerjee Bioactive compounds from algae Department of Pharmaceutical Technology, [email protected] National Institute of Pharmaceutical Education and Research, Punjab, India Y. Chisti Biodiesel from algae Institute of Technology and Engineering, Massey [email protected]. University, Private Bag 11 222, Palmerston North, New Zealand

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A APPENDIX I: CHEMICAL STRUCTURES

Name Structure Name Structure

Allenic Brominated lipid Alanine

Alginic acid Amphidinolide

4-α-acetyldictyodial Astaxanthin

Alloxanthin Allenic Fatty Acids O m n OH

130 B-C

Name Structure Name Structure

Brassicasterol Callophycoic Acid

Br Bromoform OH O H Coriolic acid OH Br Br

Bromophenol Callophycol

Canthaxathin Cellulose

131 C

Name Structure Name Structure

Cemadotin Chlorophyll a

Chlorophyll b Curacin A

Chlorophyll c1 Caulerpenyne

Chlorophyll c2 Chromene

132 C-D

Name Structure Name Structure

Cholesterol Dolastatin

6-cyano-5-methoxy- 1 2- Dehydrodldemnin B methylindolo carbazole (Aplidine where R= aldehyde)

Chitin Diadinoxanthin

OH O

Diaminopimelic acid α-Dimorphecolic acid OH

Dolabelladienetriol Diatoxanthin

133 E-I

Name Structure Name Structure

Ergosterol Glutamic acid

Flavoxanthin Glycogen

R' OH O O O Fucoxanthin Glycolipid (example) HO O O R OH H O

α-D-Galactosamine

Isoparguerol

α-D-Glucosamine

134 I-N

Name Structure Name Structure

β-Ionone Ma’iliohydrin

Isopachydictyolal Muramic acid

Kahalalide F Norharmane

Lutein

135 P

Name Structure Name Structure

Peridinin PUA - (E,E)-2,4-Decadienal

Phycocyanin Phytol

H H Polyhydroxylated O O HO HO Phycoerythrin fucophlorethol HO O O OH O O HO HO H H

Pargueol Plastoquinones

R'

Peyssonol O O O Phospholipid (example) O O O R X P O O

136 Q-Z

Name Structure Name Structure

Spheroidene Sitosterol

R'

O O Starch Triglyceride R O O R' ' O O

Synthadotin Violaxanthin

Soblidotin Warfarin

Stigmasterol

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