33

ALGAL CULTURE AND BIOTECNOLOGY WHAT ARE THE ALGAE?

The term algae is routinely used to indicate a polyphyletic (i.e., organisms that do not

share a common origin) assemblage of O2-evolving, photosynthetic organisms.

According to this definition, plants could be considered an algal division. Older

classification systems placed algae in kingdom Plantae.

Then, how to distinguish algae from plants? The answer is quite easy because the similarities between algae and plants are much

fewer than their differences.

Plants Algae

Similarities Algae and plants produce the same storage compounds, use similar

defense strategies against predators and parasites, and a strong

morphological similarity exists between some algae and plants.

Differences Plants show a very high degree of

differentiation, with roots, leaves,

stems, and xylem/phloem vascular

network.

Algae are thallophytes; do not have

roots, stems, leaves, nor well-

defined vascular tissues.

Their reproductive organs (called

archegonium and antheridium in

female and male respectively) are

surrounded by a jacket of sterile

cells.

Their reproductive structures

(called oogonium and antheridium

in female and male respectively)

consist of cells that are potentially

fertile and lack sterile cells

covering or protecting them.

They have a multicellular diploid

embryo stage (and hence the name

embryophytes is given to plants).

They do not form embryos.

Tissue-generating parenchymatous

meristems at the shoot and root

apices, producing tissues that

differentiate in a wide variety of

shapes.

Parenchymatous development is

present only in some groups.

Vascular plants are more uniform. Algae occur in dissimilar forms

such as microscopic single cell,

macroscopic multicellular loose or

filmy conglomerations, matted or

branched colonies, or more

complex leafy or blade forms,

All plants have a digenetic life cycle

with an alternation between a

haploid gametophyte and a diploid

sporophyte (the dominant stage).

Have both monogenetic and

digenetic life cycles.

OTHER DEFINITIONS

Algae are thallophytes (plants lacking roots, stems, and leaves) that have chlorophyll

a as their primary photosynthetic pigment and lack a sterile covering of cells around

the reproductive cells.

Algae are (with numerous exceptions) aquatic organisms that (with frequent

exceptions) are photosynthetic, oxygenic autotrophs that are (except for the kelps)

typically smaller and less structurally complex than land plants.

34

TECHNOLOGICAL APPLICATIONS

(MICROALGAE) Algae have a lot of technological applications which include the following:

Algae as research tools

Algae are useful laboratory organisms because they are small, grow rapidly, have

short generation times, and are easily cultivated under laboratory conditions. Cultures

of many algal species are readily available from collections maintained by experts in

several nations.

Algae as biomonitors

A bioassay is procedure that uses organisms and their responses to estimate the effects

of physical and chemical agents in the environment. Biomonitors provide early

warning of possible environmental deterioration, and may provide sensitive measures

of pollution. One common use of algal cultures is as biomonitors in the detection of

algal nutrients or substances -in natural or effluent waters- that are toxic to algae.

While daphnids and fish have traditionally been the primary biomonitor organisms in

aquatic ecosystems, algae are more sensitive than animals to some pollutants,

including detergents, textile-manufacturing effluents, dyes and especial herbicides.

Algal toxicity tests have become important components of aquatic safety assessments

for chemicals and effluents.

The use of algae for removal of mineral nutrients in wastewater effluent

Effluent treatment systems incorporate microalgal ponds, through which treated water

is channeled, just prior to discharge. The algae remove inorganic nutrients and add

oxygen.

Use of fossil algae in Paleoecological assessments

Paleoecology (also spelt palaeoecology) is to use data from fossils to reconstruct the

ecosystems of the past.

The silica skeleton of diatoms and silicoflagellates and decay-resistant cysts of

dinoflagellates persist in ocean sediments for million of years, forming records used

to deduce past environment changes.

Microalgae in animal aquaculture systems

Many marine animals cannot synthesize certain essential long-chain fatty acids in

quantities high enough for growth and survival and thus depend upon algal food to

supply them. Microalgae including Isochrysis, Pavlova, Nannochloropsis, and various

diatoms represent the primary food source for at least some stages in the life cycle of

most cultivated marine animals.

Microalgal mass cultivation for production of food, food additives, hydrocarbons

and other products

A number of microalgae are cultivated for production of human "health-foods", or

food additives such as -carotene (which is converted to vitamin A during digestion),

or protein extracts.

In general, however, the high nucleic acid content of algae limit their use in human

foods. Nucleic acids are converted to uric acid upon digestion. If consumed at a rate

more than 50 grams per day, uric acid may precipitate as sodium urate crystals in joint

cartilage, causing gout.

35

Some algae such as Spirulina are highly nutritive, full of protein and vitamins. It is

actually already available on the market in the form of tablets and pills. Spirulina

could be used in astronaut food to help ensure they keep a nutritious and balanced

diet.

Algae in space and space research

Algae are very robust organisms and are able to grow in a wide range of

environments: very high salinity, very high UV, dryness, extreme temperature or,

extreme pH. This is why algae are very good candidates for life support systems in

space or on celestial bodies.

Algae have played a significant role in space research for several decades and will

have future important applications in spacecrafts life-support systems, colonization of

Moon and Mars, and eventually in terraformation (to make it habitable) of other

planetary bodies. Experiments designed to study the growth of algae in zero-gravity

conditions of space have flown aboard rockets, satellites, the space shuttle, and the

Salyut and Mir space stations.

Algae as a sustainable source for production of renewable energy

Microalgae have long been recognized as potentially good sources for biofuel

production because of their high oil content and rapid biomass production.

Screening of algae for production of antiviral and antifungal compounds and

other pharmaceuticals

Various types of algae have proved to be sources of compounds with antibiotic and

anticancer activity, as well as pharmacologically useful products. The goal of

screening programs is to survey cultivable organisms whose medicinal properties are

unknown.

Genetic Engineering of Algae for improved technological performance

Just as crop plants are targets of genetic engineering efforts to improve productivity,

broaden environmental tolerance limits, or increase pest or pathogen resistance, so too

are useful algal species.

DNA sequences are introduced into algal cells with goal of modifying biochemical

pathways, either by changing expression of existing genes or by adding genes that

yield a new different products.

The ability to transform algal cells, i.e., to introduce and achieve desired levels of

expression of foreign genes, has been made possible by a series of technical

achievements. These include;

(1) identification of procedures that work best for incorporating foreign DNA into

algal cells;

(2) development of promoter(1)

systems for linkage to the genes of interest that

will allow expression after the DNA has been incorporated into the host

genome; and

(3) selection of an appropriate DNA marker (e.g. reporter gene)(2)

that allows the

genetic engineer to determine which cells have been transformed.

(1) A promoter is a region of DNA that initiates transcription of a particular gene.

(2) A reporter gene (often simply reporter) is a gene that researchers attach to a regulatory

sequence of another gene of interest in bacteria, cell culture, animals or plants. Reporter genes

are often used as an indication of whether a certain gene has been taken up by or expressed in

the cell or organism population.

36

4. CULTIVATION OF ALGAE

(MICROALGAE) Definition

A culture is a genetically homogenous clone propagated from one individual cell or

filament in an artificial environment.

In theory, culture conditions should resemble the alga’s natural environment as far as

possible; in reality many significant differences exist.

Basic Culturing Techniques of Microalgae

Basic principles of microbial cultivation in general are applicable to microalgae. The

unique ability of microalgae to utilize light energy, however, sets them apart from

most microorganisms. The supply of light needs to be satisfied in isolation, cultivation

and maintenance of photoautotrophic and mixotrophic microalgae.

Isolation of algae

Isolation involves the following steps:

1. Selection of sources of algae

Aquatic algae may be sampled by collecting a representative volume of water.

Algae attached to other algae, vascular plants, animals, and rocks can be collected by

scraping.

2. Enrichment of a culture

Enrichment is the process of providing a suitable environment for the growth and

reproduction of a special group of microalgae while being inhibitory or lethal for

non-target organisms.

3. Establishing Unialgal Cultures

Unialgal cultures contain only one kind of alga but may contain bacteria, fungi, or

protozoa.

To obtain a unialgal culture different techniques, some borrowed from microbiology,

are available, including:

3.1 Streaking and successive plating on agar media

Streaking is useful for single-celled, colonial, or filamentous algae that can grow on

an agar surface. Following an incubation period, morphologically distinct isolates are

picked up and transferred to a fresh agar medium, the processes can be repeated until

identical colonies are obtained on a plate.

3.2 Agar Pour Plates

Cells from the field sample or enriched culture are mixed with the non-solidified agar

and then poured into petri dishes. The non-solidified agar must be cool—only slightly

warmer than the gelling point—to avoid overheating and killing the algae. Low-

melting-point agar is preferred over normal agar.

3.3 Direct isolation Filaments can be grabbed with a slightly curved pipette tip and dragged through soft

agar (less than 1%) to remove contaminants. It is best to begin with young branches or

filament tips.

37

3.4 Single-cell isolations using Micropipette Single cells or filaments can be picked up under a dissecting microscope, using

micropipettes. The individual cells are transferred to agar medium or fresh sterile

medium for isolation.

FIGURE. An agar plate streaked with a

small, green coccoid alga, showing small

isolated colonies arising from a single cell.

FIGURE . A pipette is used to remove other small

cells (left, middle), leaving the target organism free of

contamination (right). This procedure limits the

handling of the target organism.

3.5 Atomized Cell Spray Technique A fine or atomized spray of cells can be used to

inoculate agar plates.

3.6 Serial dilution techniques

FIGURE . An illustration of the dilution technique.

3.7 Zoospores isolation Depend on isolation of zoospores immediately after they

have been released from parental cell walls, but before they stop swimming and

attached to a surface. Recently released zoospores are devoid of contaminants.

38

3.8 Gravity Separation: Centrifugation, Settling Effective method for separating

larger and heavier cells from smaller algae and bacteria.

3.9 Isolation with Use of Phototaxis. This method is effective when the algal species

exhibits phototaxis.

3.10 Isolating Epiphytes after Sonication Sonication can be employed to vibrate epiphytes from the host, and once suspended,

the epiphyte can be picked and handled.

3.11 Isolating Strongly Adhering Organisms

In this case, the cell can be washed loose with a stream of water gently blown from a

pipette (with a tube and mouthpiece). Once the stream detaches the cell from the

substrate and becomes free in the liquid, the cell can be quickly picked, rinsed, and

inoculated into the culture medium.

3.12 Isolating Sand-Dwelling Organisms The sand and water is placed in a petri dish, and the cells that move up into the

overlaying water are either collected with a micropipette or allowed to adhere to

floating coverslips.

3.13 Isolating Cysts from Sediments

The sediments can be enriched with nutrients and incubated in light so that the cysts

germinate. Once germinated, cells can be isolated with the techniques described

previously.

3.14 Removing Diatoms with Germanium Dioxide

Diatoms can be a problem for other scientists who are attempting to isolate non-

diatom algae because they grow very quickly, and can often outcompete other algae.

In most cases, the addition of germanium dioxide to the culture leads to the death of

most or all diatoms. *************************************************

PRODUCING AXENIC CULTURES

While ―Unialgal‖ cultures contain only one kind of alga but, may contain bacteria,

fungi, or protozoa, AXENIC cultures contain only one alga without bacteria, fungi,

protozoa or any other living organism.

Biological contamination of algal cultures by other eukaryotes and/or prokaryotic

organisms in most cases

invalidates experimental work, and

may lead to the extinction of the desired algal species in culture through

outcompetition or grazing.

In practice, it is very difficult to obtain bacteria-free (axenic) cultures, and although

measures should be taken to minimize bacterial numbers, a degree of bacterial

contamination is often acceptable.

Although sterile cultures of microalgae may be obtained from specialized culture

collections, some of the basic purification techniques are described below.

1. Extensive cell washing

Under a dissecting microscope, an individual algal cell is picked up using a

micropipette and placed in a sterile liquid medium. The organism is then transferred

through a series of sterile media.

39

2. Density gradient centrifugation

Used to separate algae from bacteria. Silica sol, percoll has is used to produce density

gradient for the separation of microalgae, where each species is separated into a sharp

band. The algae at a particular position within the gradient are collected by

fractionation of the gradient.

3. UV irradiation

Most algae are slightly more resistant to ultraviolet light than bacterial cells. Thus,

following UV irradiation, washing and diluting a sample, then spraying or streaking it

on selective agar medium may produce pure algal culture free of bacteria.

4. Filtration

Algae can be separated from bacteria using membrane filters. Sonication is often

employed to break up the algae into small length filaments (in case of filamentous

algae) and to detach adhering bacteria. The diluted sample is then vacuum filtered.

5. Antibiotics

Antibiotics discourage growth of contaminating bacteria and cyanobacteria in algal

culture, For example:

Imipenem (110 µgml_1

) was used to purify unicellular eukaryotic microalgae.

The antibiotics nystatin (100 µgml_1

) and cycloheximide (100 µgml_1

) were used

to eliminate fungal contaminants.

Best results appear to occur when an actively growing culture of algae is exposed

to a mixture of penicillin, streptomycin, and gentamycin for around 24 h.

Other antibiotics that can be used include chloramphenicol, tetracycline, and

bacitracin..

Different algal species tolerate different concentrations of antibiotics, so a range of

antibiotic concentrations should be used and then select the culture that has surviving

algal cells but no surviving bacteria or other contaminants. Antibiotic solutions should

be made with distilled water and filter-sterilized (0.2 µm filter units) into sterile tubes,

and should be stored frozen until use.

6. Bleach Resistant stages such as zygotes or akinetes can be treated with bleach to kill other

organisms, then planted on agar for germination. It is usually necessary to try several

different concentrations of bleach and times of exposure to find a treatment that will

kill other organisms without harming the alga.

Checking the Sterility of cultures

Sterility of cultures should be checked by microscopic examination (phase contrast)

or by adding a small amount of sterile bacterial culture medium (e.g., 0.1% peptone)

to a microalgal culture, incubated in dark and observing regularly for two to three

days for bacterial growth. There should be no indications of any microbial growth.

40

CULTURE PARAMETERS

A culture has three distinct components:

(1) a culture medium contained in a suitable vessel;

(2) the algal cells growing in the medium;

(3) air, to allow exchange of carbon dioxide between medium and atmosphere.

For an entirely autotrophic alga, all that is needed for growth is light, CO2, water,

nutrients, and trace elements. By means of photosynthesis the alga will be able to

synthesize all the biochemical compounds necessary for growth. Only a minority of

algae is, however, entirely autotrophic; many are unable to synthesize certain

biochemical compounds (certain vitamins, e.g.) and will require these to be present in

the medium (obligate mixotropy).

The most important parameters regulating algal growth are nutrient quantity and

quality, light, pH, turbulence, salinity, and temperature. The most optimal parameters

as well as the tolerated ranges are species specific and the parameter that is optimal

for one set of conditions is not necessarily optimal for another.

Temperature

The temperature at which cultures are maintained should ideally be as close as

possible to the temperature at which the organisms were collected. Most commonly

cultured species of microalgae tolerate temperatures between 16 and 27C. An

intermediate value of 18–20C is most often employed. Temperatures lower than

16C will slow down growth, whereas those higher than 35

C are lethal for a number

of species.

Light

Light is the source of energy which drives photosynthetic reactions in algae and in

this regard intensity, spectral quality, and photoperiod need to be considered.

Light intensity plays an important role, but the requirements greatly vary with the

culture depth and the density of the algal culture: at higher depths and cell

concentrations the light intensity must be increased to penetrate through the

culture. Too high light intensity (e.g., direct sunlight, small container close to

artificial light) may result in photoinhibition. Moreover, overheating due to both

natural and artificial illumination should be avoided.

Spectral quality Light may be natural or supplied by fluorescent tubes.

Fluorescent tubes emitting either in the blue or the red light spectrum can be used,

as these are the most active portions of the light spectrum for photosynthesis.

Light intensity and quality can be manipulated with filters.

Photoperiod Many microalgal species do not grow well under constant

illumination, and hence a light/dark (LD) cycle is used (maximum 16:8 LD,

usually 14:10 or 12:12).

PH

The pH range for most cultured algal species is between 7 and 9, with the optimum

range being 8.2–8.7, though there are species that dwell in more acid/basic

environments. Complete culture collapse due to the disruption of many cellular

processes can result from a failure to maintain an acceptable pH. The latter is

accomplished by aerating the culture. In the case of high-density algal culture, the

addition of carbon dioxide allows to correct for increased pH, which may reach

limiting values of up to pH 9 during algal growth.

41

Salinity

Marine algae are extremely tolerant to changes in salinity. Most species grow best at a

salinity that is slightly lower than that of their native habitat, which is obtained by

diluting sea water with tap water. Salinities of 20–24 g l-1

(part per thousand, ppt, ‰)

are found to be optimal.

Mixing

Mixing is necessary

to prevent sedimentation of the algae,

to ensure that all cells of the population are equally exposed to the light and

nutrients,

to avoid thermal stratification (e.g., in outdoor cultures),

to improve gas exchange between the culture medium and the air. For very dense

cultures, the CO2 originating from the air (containing 0.03% CO2) bubbled

through the culture is limiting the algal growth and pure carbon dioxide may be

supplemented to the air supply (e.g., at a rate of 1-5% of the volume of air). CO2

addition furthermore buffers the water against pH changes as a result of the

CO2/HCO-3 balance.

It should be noted that in the ocean cells seldom experience turbulence, and hence

mixing should be gentle.

Culture vessels

Culture vessels should have the following properties:

non-toxic (chemically inert);

reasonably transparent to light;

easily cleaned and sterilized; and

provide a large surface to volume ratio (depending on the organism).

MEDIA CHOICE AND PREPARATION

When choosing a culture medium, the natural habitat of the species in question

should be considered in order to determine its environmental requirements. Media can

be classified as being defined or undefined.

Defined media, which are often essential for nutritional studies, have constituents that

are all known.

Undefined media, on the other hand, contain one or more natural or complex

ingredients where, the composition of which is unknown and may vary, such as, agar,

soil extract or natural seawater,

Media may be prepared by combining concentrated stock solutions, which are not

combined before use, to avoid precipitation and control contamination. Reagent grade

chemicals and bidistilled (or purer) water should be used to make stock solutions of

enrichments. Gentle heating and/or magnetic stirring of stock solutions can be used to

ensure complete dissolution.

When preparing a stock solution containing a mixture of compounds (such as the

trace metal solution of for example F/2 medium), each compound has to be dissolved

individually in a minimal volume of water before mixing, then combined with the

other and the volume diluted to the needed amount. Stock solution should be stored at

low temperature in tightly sealed glassware, because evaporation may alter initial

concentrations.

42

MARINE MEDIA

Seawater is an ideal medium for growth of marine species, but it is an intrinsically

complex medium, containing over 50 known elements in addition to a large but

variable number of organic compounds.

Media for the culture of marine phytoplankton consist of a seawater base which may

be supplemented by various substances essential for algal growth, including nutrients,

trace metals and chelators, vitamins, soil extract, and buffer compounds.

The salinity of the seawater base should first be checked (30–35‰ for marine

phytoplankton), and any necessary adjustments (addition of fresh water/evaporation)

made before addition of enrichments.

Seawater, stock solutions of enrichments, and the final media must be sterile in order

to prevent (or more realistically minimize) biological contamination of unialgal

cultures.

Autoclaving is a process which has many effects on seawater and its constituents:

Potentially altering or destroying inhibitory organic compounds, as well as

beneficial organic molecules.

Because of the steam atmosphere in an autoclave, CO2 is driven out of the

seawater and the pH is raised to about 10, a level which can cause

precipitation of the iron and phosphate added in the medium. Some of this

precipitate may disappear upon

(1) re-equilibriation of CO2 on cooling.

(2) The presence of ethylenediaminetetraacetic acid (EDTA) and the use of

organic phosphate may reduce precipitation effects.

(3) Addition of 5% or more of distilled water may also help to reduce

precipitation (but may affect final salinity).

(4) The best solution, however, if media are to be autoclaved, is to sterilize iron

and phosphate (or even all media additions) separately and add them

aseptically afterwards.

Agar medium

Some marine microalgae grow well on solid substrate. A 3% high grade agar can be

used for the solid substrate. The agar and culture medium however should not be

autoclaved together, because toxic breakdown products can be generated. The best

procedure is to autoclave 30% agar in deionized water in one container and nine times

as much seawater base in another. After removing from the autoclave, sterile nutrients

are added aseptically to the water, which is then mixed with the molten agar. After

mixing, the warm fluid is poured into sterile Petri dishes, where it solidifies when it

cools.

Seawater

The quality of water used in media preparation is very important.

1. Natural seawater can be collected near shore, but its salinity and quality is often

quite variable and unpredictable, due to anthropogenic pollution, toxic metabolites

released by algal blooms in coastal waters.

The quality of coastal water may be improved by

ageing for a few months at 4C (allowing bacteria degradation),

by autoclaving (heat may denature inhibitory substances),

or by filtering through acid-washed charcoal (which absorbs toxic organic

compounds).

Filtering through filter papers.

43

The low biomass and continual depletion of many trace elements from the surface

waters of the open ocean by biogeochemical processes makes this water much

cleaner, and therefore preferable for culturing purposes. Seawater should be stored in

cool dark conditions.

2. Artificial seawater, made by mixing various salts with deionized water. Artificial

or synthetic seawater has the advantage of being entirely defined from the chemical

point of view, but it is very laborious to prepare, and often does not support

satisfactory algal growth. Trace contaminants in the salts used are at rather high

concentrations in artificial seawater because so much salt must be added to achieve

the salinity of full strength seawater.

3. Commercial preparations are available, which consists of synthetic mixes of the

major salts present in natural sea water, such as Tropic Marine Sea Salts produced in

Germany for Quality Marine (USA) and Instant Ocean Sea Salts by Aquarium System

(USA).

Nutrients

The term ‘nutrient’ is applied to a chemical required in relatively large quantities, but

can be used for any element or compound necessary for algal growth.

The average concentrations of constituents of potential biological importance found in

typical seawater are summarized in Table 6.10.

These nutrients can be divided into three groups, I, II, and III; with decreasing

concentration:

Group I. Concentrations of these constituents exhibit essentially no variation in

seawater, and high algal biomass cannot deplete them in culture media. These

constituents do not, therefore, have to be added to culture media using natural

seawater, but do need to be added to deionized water when making artificial

seawater media.

Group II. Also have quite constant concentrations in seawater. Because

microalgal biomass cannot deplete their concentrations significantly, they also do

not need to be added to natural seawater media. Standard artificial media (and

some natural seawater media) add molybdenum (as molybdate), and/or selenium

(as selenite), which has been demonstrated to be needed by some algae.

Strontium, bromide, and flouride, all of which occur at relatively high

44

concentrations in seawater and none of which have been shown to be essential for

microalgal growth.

Group III. All known to be needed by microalgae (silicon is needed only by

diatoms and some chrysophytes, and nickel is only known to be needed to form

urease when algae are using urea as a nitrogen source). These nutrients are

generally present at low concentrations in natural seawater, and because

microalgae take up substantial amounts, concentrations vary widely. All of these

nutrients (except silicon and nickel in some circumstances) generally need to be

added to culture media in order to generate significant microalgal biomass.

45

Nitrate is the nitrogen source most often used in culture media, but ammonium can

also be used, and indeed is the preferential form for many algae because it does not

have to be reduced prior to amino acid synthesis. Ammonium concentrations greater

than 25 µM are, however, often reported to be toxic to phytoplankton, so

concentrations should be kept low.

Inorganic (ortho) phosphate, the phosphorus form preferentially used by

microalgae, is most often added to culture media, but organic (glycero) phosphate is

sometimes used, particularly when precipitation of phosphate is anticipated (when

nutrients are autoclaved in the culture media rather than separately). Most microalgae

are capable of producing cell surface phosphatases, which allow them to utilize this

and other forms of organic phosphate as a source of phosphorus.

The trace metals that are essential for microalgal growth are incorporated into

essential organic molecules, particularly a variety of coenzyme factors that enter into

photosynthetic reactions. Of these metals, the concentrations of Fe, Mn, Zn, Cu and

Co (and sometimes Mo and Se) in natural waters may be limiting to algal growth.

Chelators are chemical molecules that complex with metals and influence the

biological availability of these metals. Chelators act as trace metal buffers,

maintaining constant concentrations of free ionic metal. Without proper chelation some metals (such as Cu) are often present at toxic

concentrations, and others (such as Fe) tend to precipitate and become unavailable to

phytoplankton.

In natural seawater, dissolved organic molecules (generally present at concentrations

of 1–10 mg l-1

) act as chelators. The most widely used chelator in culture media

additions is EDTA, which must be present at high concentrations because most

complexes with Ca and Mg, present in large amounts in seawater. EDTA may have an

additional benefit of reducing precipitation during autoclaving. High

concentrations have, however, occasionally been reported to be toxic to microalgae.

As an alternative the organic chelator citrate is sometimes utilized.

Vitamins

Roughly all microalgal species tested have been shown to have a requirement for

vitamin B12 (cobalamin), which appears to be important in transferring methyl groups

and methylating toxic elements such as arsenic, mercury, tin, thallium, platinum, gold,

and tellurium, around 20% need thiamine (vitamin B1), and less than 5% need biotin

(vitamin H). It is recommended that these vitamins are routinely added to culture

media.

Soil Extract

Soil extract has historically been an important component of culture media. The

solution provides macronutrients, micronutrients, vitamins, and trace metal chelators

in undefined quantities, each batch being different, and hence having unpredictable

effects on microalgae. With increasing understanding of the importance of various

constituents of culture media, soil extract is less frequently used.

Buffers

The control of pH in culture media is important because

certain algae grow only within narrowly defined pH ranges

in order to prevent the formation of precipitates.

46

Except under unusual conditions, the pH of natural seawater is around 8. Because of

the large buffering capacity of natural seawater due to its bicarbonate buffering

system it is quite easy to maintain the pH of marine culture media. The buffer system

is overwhelmed only

during autoclaving, when high temperatures drive CO2 out of solution and

hence cause a shift in the bicarbonate buffer system and an increase in pH, or

in very dense cultures of microalgae, when enough CO2 is taken up to produce

a similar effect.

As culture medium cools after autoclaving, CO2 reenters solution from the

atmosphere, but certain measures must be taken if normal pH is not fully restored:

The pH of seawater may be lowered prior to autoclaving (adjustment to pH 7–

7.5 with 1 M HCl) to compensate for subsequent increases.

Certain media recipes include additions of extra buffer, either as bicarbonate,

Tris (Trishydroxymethyl-aminomethane), or glycylglycine to supplement the

natural buffering system.

o Tris has occasionally been cited for its toxic properties to microalgae

such as Haematococcus sp.

o Glycylglycine is rapidly metabolized by bacteria and hence can only be

used with axenic cultures.

These additions are generally not necessary if media are filter sterilized,

unless very high cell densities are expected.

The problem of CO2 depletion in dense cultures may be reduced by

o having a large surface area of media relative to the volume of the

culture, or by

o bubbling with either air (CO2 concentration ca. 0.03%) or air with

increased CO2 concentrations (0.5–5%).

Unless there is a large amount of biomass taking up the CO2, the higher

concentrations could actually cause a significant decline in pH.

When bubbling is employed, the gas must first pass through an inline 0.2 µm

filter unit (e.g., Millipore Millex GS) to maintain sterile conditions. For many

microalgal species, aeration is not an option because the physical disturbance

may inhibit growth or kill cells.

Table 6.18 summarizes the algal classes that have been successfully cultured and their

corresponding media names.

For a full range of possible media refer to the catalog of strains from culture

collections present all over the world such as:

UTEX (Culture Collection of Algae at the University of Texas at Austin,

http://www.bio.utexas.edu/research/utex/)

CCAP (Culture Collection of Algae and Protozoa, Argyll, Scotland,

http://www.ife.ac.uk/ccap/)

UTCC (University of Toronto Culture Collection of Algae and Cyanobacteria,

http://www.botany.utoronto.ca/utcc/)

CCMP (The Provasoli-Guillard National Center for Culture of Marine

Phytoplankton, Maine, http://ccmp.bigelow.org/)

CSIRO (CSIRO Collection of living microalgae, Tasmania,

http://www.marine.csiro.au/ microalgae/collection.html).

47

CULTURE METHODS

Algae can be produced according to a great variety of methods. The terminology used

to describe the type of algal culture include:

Indoor/Outdoor. Indoor culture allows control over illumination, temperature,

nutrient level, contamination with predators and competing algae, whereas

outdoor algal systems though cheaper, make it very difficult to grow specific algal

cultures for extended periods.

Open/Closed. Open cultures such as uncovered ponds and tanks (indoors or

outdoors) are more readily contaminated than closed culture vessels such as tubes,

flasks, carboys, bags, etc.

Axenic (=sterile)/Xenic. Axenic cultures are free of any foreign organisms such

as bacteria, but this cultivation is expensive and difficult, because it requires a

strict sterilization of all glassware, culture media and vessels to avoid

contamination. These constraints make it impractical (and very expensive) for

commercial operations. On the other hand, non-axenic or xenic cultivation,

though cheaper and less laborious, are more prone to crash, less predictable, and

often of inconsistent quality.

Batch, Continuous, and Semi-Continuous. These are the three basic types of

algal culture which will be described in the following sections.

BATCH CULTURES

This is the most common method for cultivation of microalgal cells. The batch culture

consists of a single inoculation of cells into a container of fertilized seawater followed

by a growing period under favorable conditions for several days and finally harvesting

when the algal population reaches its maximum or near-maximum density.

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Batch culture systems are widely applied because of their advantages which include:

Ease of operation and simplicity

Flexibility, allowing to change species

allowing to remedy defects in the system rapidly.

Although often considered as the most reliable method, batch culture is not

necessarily the most efficient method.

Disadvantages

Batch cultures are harvested just prior to the initiation of the stationary phase and

must thus always be maintained for a substantial period of time.

The quality of the harvested cells may be less predictable than that in continuous

systems and for example vary with the timing of the harvest.

Another disadvantage is the need to prevent contamination, especially, during the

initial inoculation and early growth period. Because the density of the desired

microalgae is low and the concentration of nutrients is high, any contaminant with

a faster growth rate is capable of outgrowing the culture.

Batch cultures also require a lot of labor to harvest, clean, sterilize, refill, and

inoculate the containers.

Population dynamics

Batch culture systems can be described by different phases, reflecting changes in the

biomass and in its environment.

Algal population shows a typical pattern of growth according to a sigmoid curve

(Figure ), consisting of a succession of six phases, characterized by variations in the

growth rate; the six phases are summarized in Table 6.19.

Time

FIGURE Growth curve of an algal population under batch culture conditions.

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The key to the success of algal production is maintaining all cultures in the

exponential phase of growth. Also, the nutritional value of the produced algae is

inferior once the culture is beyond Phase 4 due to

1. reduced digestibility,

2. deficient composition,

3. and possible production of toxic metabolites.

Upscaling cultures

The upscaling of microalgae production starts from small containers (0.5 ml) and

proceeds through various steps up to the mass production in Polyethylene bags (up to

450 liter). Each step involves an increase of the culture volume: when mature, the

algal population of a smaller volume is sacrificed to replicate the same vessel and to

inoculate larger vessels. According to the algal concentration, the volume of the

inoculums amounts to 2-10% of the final culture volume.

The following consecutive stages might be utilized: test tubes, 2 l flasks, 5 and 20 l

carboys, 160 l cylinders, 500 l indoor tanks, 5,000 l to 25,000 l outdoor tanks.

Fig A typical scheme of a batch type production

CONTINUOUS CULTURES

In continuous cultures, cultures are maintained at a chosen point on the growth curve

by the regulated addition of fresh culture medium. In practice, a volume of fresh

culture medium is added automatically at a rate proportional to the growth rate of the

alga, while an equal volume of culture is removed. This method of culturing algae

permits the maintenance of cultures very close to the maximum growth rate, because

the culture never runs out of nutrients.

Two categories of continuous cultures can be distinguished:

Turbidostat culture, in which fresh medium is delivered only when the cell

density of the culture reaches some predetermined point.

Chemostat culture (constant chemical environment), in which a flow of fresh

medium is introduced into the culture at a steady, predetermined rate, in this way

the growth rate is kept constant. In many chemostat continuous culture systems,

the rate of media flow is often set at approximately 20% of culture volume per

day.

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Fig. . Schematic diagram of a chemostat setup.

Advantages of continuous cultures

Continuous cultures have the advantages of producing algae of more predictable

quality.

They are amenable to technological control and automation, which in turn

increases the reliability of the system and reduces the need for labor

Disadvantages of continuous cultures The disadvantages of the continuous system are

Its relatively high cost and complexity.

The requirements for constant illumination and temperature mostly restrict

continuous systems to indoors and this is only feasible for relatively small

production scales.

SEMI-CONTINUOUS CULTURES

In a semi-continuous system the fresh medium is delivered to the culture all at once

and spent culture flows out into a collecting vessel. The culture is allowed to regrow

(for 24 h as example), then the procedure is repeated. As the culture is not harvested

completely, the semicontinuous method yields more algae than the batch method for

a given tank.

51

COMMERCIAL-SCALE CULTURES

Cultivation of microalgae for commercial purposes is presently carried out, with only

a few exceptions, in open systems. A number of types of ponds have been designed

and experimented with for microalgae cultivation. They vary in size, shape, material

used for construction, type of agitation and inclination.

1. Circular ponds

2. Raceway ponds

3. Inclined systems (sloping cascade)

Fig. Illustration of algal mass culture systems

Nutrient medium for large scale outdoor cultures

The nutrient medium for outdoor cultures is based on that used indoors, but

agricultural-grade fertilizers are used (nitrate and phosphate fertilizers and a few

other micronutrients) instead of laboratory-grade reagents. Cultures from indoor

production may serve as inoculum for monospecific cultures.

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Outdoor advantages/disadvantages

Advantages Algal production in outdoor ponds are easier and inexpensive to build.

Disadvantages

Algae cannot be maintained for prolonged period

Outdoor culture is only suitable for a few, fast-growing species due to

problems with contamination by predators, parasites, and more opportunistic

algae that tend to dominate regardless of the species used as inoculum.

Outdoor production is often characterized by a poor batch to batch consistency

Unpredictable culture crashes could be caused by changes in weather,

sunlight, or water quality.

At present, large-scale commercial production of microalgae biomass is limited to

Dunaliella, Haematococcus, Arthrospira, and Chlorella, which are cultivated in open

ponds at farms located around the world. These algae are a source for viable and

inexpensive carotenoids, pigments, proteins, and vitamins that can be used for the

production of nutraceuticals, pharmaceuticals, animal feed additives, and cosmetics.

Lakes and natural ponds

When microalgae find suitable climatic conditions and sufficient nutrients, they grow

profusely. If the chemical characteristics of the water are selective, for example, due

to high pH or high salinity, the bloom is almost monospecific. There are many

examples of eutrophic lakes or small natural basins exploited for microalgae

production.

Fig . (a) A Kanembu woman harvesting Arthrospira platensis from lake Kossorom, a permanent

alkaline lake at the north-east fringe of lake Chad (Chad).; (b) Harvesting of Arthrospira from an old

volcanic crater filled with alkaline water in Myanmar.

53

PHOTOBIOREACTORS (PBR)

Definition of photobioreactors

Photobioreactors can be defined as closed culture systems for phototrophs in which a

great proportion of the light (>90%) has to pass through the transparent reactor’s

walls to reach the cultivated cells.

Advantages

PBR do not allow, or strongly limit, direct exchange of gases and contaminants

(dust, microorganisms, etc.) between the culture and the atmosphere.

These devices provide a protected environment for cultivated species, relatively

safe from contamination by other microrganisms,

culture parameters such as pH, oxygen and carbonic dioxide concentration, and

temperature can be better controlled, and provided in known amount.

Moreover, they prevent evaporation and reduce water use,

lower CO2 losses,

permit higher cell concentration, thus reducing operating costs, and attain higher

productivity.

Disadvantages, these systems are more expensive to build and operate, due to the

need of cooling, strict control of oxygen accumulation, and biofouling, and their use

must be limited to the production of very high-value compounds from algae that

cannot be cultivated in open ponds.

Classification of photobioreactors

Photobioreactors can be classified on the basis of both design and mode of operation.

In design terms, the main categories of reactors are:

(1) flat or tubular;

(2) horizontal, inclined, vertical or spiral;

(3) serpentine or manifold.

An operational classification of PBR would include

(4) air or pump mixed and

(5) single-phase reactors (filled with media, with gas exchange taking place in a

separate gas exchanger), or two-phase reactors (in which both gas and liquid are

present and continuous gas mass transfer takes place in the reactor itself).

Construction materials provide additional variation and subcategories; for example

(6) glass or plastic and

(7) rigid or flexible PBR.

Axenic PBR are reactors operated under sterile conditions. Although a major

characteristic of PBR is their ability to limit contamination, it must be made clear that

an effective barrier, and thus operation under truly sterile conditions, is not achieved

except in the few special designs developed expressly for that purpose.

54

I. Tubular photobioreactors

1. Serpentine photobioreactors

Fig. . (a) Serpentine reactors at the Department of Chemical Engineering of the University of Almeria

(Spain); (b) A two-plane serpentine reactor at the Centro di Studio dei Microrganismi Autotrofi of the

CNR (Florence, Italy).

2. Manifold photobioreactors

In manifold PBR, a series of parallel tubes are connected at the ends by two

manifolds, one for distribution and one for collection of the culture suspension.

55

4. Helical photobioreactors (bio-coil PBR)

Helical PBR consist of small-diameter, generally flexible tubes wound around an

upright structure.

II. Flat photobioreactors

III. Vertical cylinders and sleeves

Fig. . (a) Polyethylene sleeves at the Institute for Applied Research (Beer-Sheva); (b) Annular

columns at the Department of Agricultural Biotechnology of the University of Florence (Italy).

56

DESIGN CRITERIA FOR PHOTOBIOREACTORS

Design criteria for PBR should aim at achieving high productivities and high

efficiency of conversion of light energy, providing, at the same time, the necessary

reliability and stability to the cultivation process by means of a cost-effective culture

system. An efficient PBR can not be properly designed without adequate knowledge

of the physiology in mass culture of the organisms to be cultivated. Since

phototrophic microorganisms are highly diverse in their morphology, nutritional and

light requirements, and resistance to stresses, PBR can not be designed so as to adapt

to all organisms and all conditions. The main design criteria for PBR include:

1. Surface-to-volume ratio (s/v)

The ratio between the illuminated surface area of a reactor and its volume (s/v)

determines the amount of light that enters the system per unit volume. Generally, the

higher the s/v, the higher the cell concentration at which the reactor can be operated

and the higher the volumetric productivity of the culture. There is an optimal s/v at

which maximum productivity per unit of illuminated surface area is obtained.

2. Orientation and inclination

Generally, sun-oriented systems (south-facing and tilted so as to intercept maximum

solar radiation) achieve higher volumetric productivities.

3. Oxygen accumulation

Oxygen concentrations equivalent to four or five times saturation with respect to air,

is toxic to many phototrophs, and thus requires frequent degassing.

4. Mixing

Mixing is necessary to prevent cells from settling, to avoid thermal stratification, to

distribute nutrients, to remove photosynthetically generated oxygen and to ensure that

cells experience alternating periods of light and darkness of adequate length.

5. Temperature control

Maximum productivity can be achieved only at the optimal temperature for growth.

PBR generally require cooling at midday. Shading, immersion in a water bath and

water spraying are the most common solutions adopted to avoid overheating of PBR

outdoors.

6. Supply of carbon dioxide

The residence time of the injected CO2 bubbles should be sufficient for complete

absorption.

7. Materials

Materials for PBR must lack toxicity have high transparency, high mechanical

strength, of high durability (resistance to weathering), chemical stability and low cost.

Ease of cleaning is also another important operational issue.

Quantitative determination of algal density and growth

1. Cell count

2. Dry and wet mass

3. Protein concentration determination

4. Chlorophyll determination

5. Total organic carbon (TOC) measurement

57

Mariculture of Seaweeds

Macroscopic marine algae form an important living resource of the oceans. Seaweeds

are food important for humans and animals, as well as fertilizers for plants and a

source of various chemicals. Seaweeds have been gaining momentum as a new

experimental system for biological research and as an integral part of integrated

aquaculture systems. We all use seaweed products in our day-to-day life in some way

or other.

For example, some seaweed polysaccharides are used in toothpaste, soap, shampoo,

cosmetics, milk, ice cream, meat, processed food, air freshener, and many other items.

In many oriental countries such as Japan, China, Korea, and others, seaweeds are diet

staples.

During the last 50 years, approximately 100 seaweed taxa have been tested in field

farms, but only a dozen are being commercially cultivated today. Table 1 provides

production data for the top taxa.

TABLE 1 Top cultivated seaweed genera in the world during 2000 (FAO 2003).

Cultivation methods

Different taxa require different farming methods. Although some seaweeds need one-

step farming through vegetative propagation, others need two-step or multistep

farming. The latter must be propagated from spores and cannot survive if propagated

vegetatively.

IN VEGETATIVE CULTIVATION, small pieces of seaweed are taken and placed in an

environment that will sustain their growth. When they have grown to a suitable size

they are harvested, either by removing the entire plant or by removing most of it but

leaving a small piece that will grow again. When the whole plant is removed, small

pieces are cut from it and used as seedstock for further cultivation. The seaweed

should be held in its environment in several ways (Fig. 1).

58

FIGURE 1. (a) Rocky substratum with attached seaweed being transplanted to a new site. (b) seaweed attached to rocks with rubber bands; method used for anchoring transplants in soft sediments. (c) Plants

inserted with a fork directly into soft sediments. (d) Plants attached to sand-filled plastic tubes. (e)

seaweed attached to a rope, which is stretched between two poles pushed into the sediments. (f )

Attachment of plants for rope culture. (g) Bamboo floating frame with seaweed attached to ropes

spanning the frame. (h) A floating net with seaweed attached to the meshes.

The suitable environment varies among species, but must meet requirements for

salinity of the water, nutrients, water movement, water temperature and light.

59

CULTIVATION INVOLVING A REPRODUCTIVE CYCLE, with alternation of generations,

is necessary for many seaweeds; for these, new plants cannot be grown by taking

cuttings from mature ones. This is typical for many of the seaweeds.

SEAWEED CULTIVATION IN THE SEA

Cultivation occurs along coastal areas. To hold the seaweeds in place, a variety of

farm structures are used. These include long lines to which ropes with seaweeds are

attached, nets stretched out on frames, and ropes supported by poles. Details of these

different systems are provided in the following sections.

1. Long-Line Cultivation Systems

This system is developed by Chinese for cultivating Laminaria. In this method,

sporelings (―seedlings‖) are produced in artificially cooled water at 8-10 ºC in

greenhouses and later planted out in the ocean. For cultivation, Laminaria must go

through its life cycle, and this involves an alternation of generations between a large

sporophyte - the sporophyte is what is harvested as seaweed- and a microscopic

gametophyte.

Fig. 2 A Chinese long-line farm for cultivation of Laminaria: The long line is anchored by a rope at

each end and held afloat by buoys. Culture lines with attached Laminaria include a spacing segment at

the top and bottom of the 2- to 5-m-long rope and weighed. The anchor ropes are held by stakes or

concrete weights.

The Laminaria is harvested in 6-7 months when it is 3-4 metres in length. By the

same method, the Japanese also farm a similar kelp, Undaria, which is sold as the

food wakame.

60

FIGURE 3.. (d) Long line with Laminaria after 8 months of growth (Yellow Sea, China). (e) Long line

showing attached Laminaria plants (South Korea). (f ) Young sporophytes growing on long line.

2. Net-Style Farm Systems

Porphyra, or nori, is the world's most valuable marine crop. The crop is valued at

more than 1 billion dollars worldwide and is used mainly for human food. These

plants grow mainly in cooler waters. Porphyra reproduces by both sexual and asexual

modes of reproduction.

Nori grows as a flat sheet. Porphyra are allowed to grow to 15–30 cm in about 40–50

days before they are harvested (Fig. 5 c,d). The remaining thalli are allowed to grow

and may be ready for a second harvest after another 15 to 20 days. The harvested crop

is washed and transferred to an automatic nori-processing machine, which cuts the

blades into small pieces. The nori is then processed by the cultivator into dried

rectangular sheets or processed by the manufacturer per market requirements.

Fig. 4 A Japanese Porphyra culture net system: Poles hold the net in

place so that it is exposed at low tides. The Porphyra grows attached to the nets.

61

3. Line and Rope Farm Systems

Farmers in the Philippines use rope and lines to grow tropical seaweeds. These

seaweeds comprise species of genera Eucheuma and Kappaphycus. Plants are

propagated vegetatively, the seaweeds are tied to the lines, and the lines are then

staked over the bottom. Eucheuma and Kappaphycus are important carrageenophytes.

FIGURE 6. (a) Rope cultivation of Gracilaria using monofilament long lines. (b) Enlarged image

showing a Gracilaria plant held within the twists of the lone line rope. (c) Eucheuma farmers

harvesting plants from submerged long lines. (d) Open water cultivation and harvesting of

Kappaphycus; diver bringing a large plant to the boat. Eucheuma and Kappaphycus are tied to the lines

that are held just above the sediment surface. The depth of the plants are adjusted so that they are not

exposed at low tides. (e) Typical outrigger boat used for open water harvesting.

4. Offshore or Deep-Water Seaweed Cultivation

Various attempts were made to cultivate the brown alga Macrocystis pyrifera on farm

structures designed for use in deep water. The nutrients for these seaweeds were to

come from cold, deep waters that would be pumped up into the growing kelps. The

benefits of this approach is that growth of macroalgae would not be constrained by

available shallow nearshore regions.

LAND-BASED CULTIVATION SYSTEMS

TANKS Tank systems may hold promise for the processing of polluted water.

PONDS Macroalgae can also be grown in ponds.