Mass Cultivation of Freshwater Microalgae

J Masojdek, Institute of Microbiology, Trebon, Czech Republic; University of South Bohemia, Ceske Budejovice, Czech Republic
G Torzillo, Istituto per lo Studio degli Ecosistemi, CNR, Sesto Fiorentino, Italy
2014 Elsevier Inc. All rights reserved.

Introduction
Biological Principles and Technology of Mass Cultivation
Light
Temperature
Culture Monitoring and Maintenance
Cultivation Systems
Laboratory Cultivation
Open Outdoor Systems
Closed and Semiclosed Outdoor Photobioreactors
Heterotrophic Fermenters
Biotechnologically Important Strains of Microalgae
Arthrospira (Spirulina)
Chlorella
Dunaliella
Haematococcus
Microalgae for Aquaculture
Processing of Microalgal Biomass
Exploitation of Microalgal Products
Nutrition
High-value and Bioactive Compounds
Microalgae for Biofuels
Ecological Application of Microalgal Mass Cultures
Wastewater Treatment
Future Developments and Prospects
Acknowledgements
References

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Introduction
In applied phycology, the term microalgae is generally used in its broadest sense to mean both prokaryotic cyanobacteria and
eukaryotic microscopic algae. These organisms are truly ubiquitous since they inhabit all ecosystems from cold, polar regions
through to extremely alkaline or saline habitats, from hot springs to arid soils. Cyanobacteria, in particular, represent the oldest
group of organisms that began the creation of the Earths oxygen-carrying atmosphere almost 3 billion years ago. Microalgae also
represent important CO2 consumers and primary producers being the basis of the food chain in aquatic environments.
Furthermore, they represent one of the most efficient converters of solar energy to biomass.
In nature, water blooms develop in eutrophic (nutrient-rich) reservoirs where phytoplankton populations are only occasionally
mixed by wind or current. In these situations, typical concentrations of algal biomass are much below 1 g of dry matter per liter. For
centuries, natural blooms of the cyanobacterium Spirulina (now referred to as Arthrospira) were harvested in certain environments
of alkaline soda lakes in countries such as Chad, Mexico, or Myanmar, and used as a food supplement. By contrast, the present
nutrient enrichment of surface waters, the results of human activity such as human waste, industrial effluents, agricultural fertilizers
and aquaculture farming, has caused massive developments of dangerous microalgal blooms; these represent an ecological threat
due to their potential to seriously reduce water quality and create hazardous environmental problems.
In microalgal biotechnology, suitable species can be grown as productive strains in aquacultures facilitating efficient manipulation of cultivation process. Although many microalgal strains are cultivated worldwide for different purposes, the bulk of annual
biomass production is represented by only four species: the cyanobacterium Spirulina and the green microalgae Chlorella, Dunaliella
and Haematococcus. Dense, well-mixed mass culture of microalgae (>0.5 g biomass per liter) with sufficient nutrition and gas

Change History: June 2014. J Masojdek and G Torzillo updated the sections on Light, Culture Monitoring and Maintenance, Cultivation Systems,
Heterotrophic Fermenters, Microalgae for Aquaculture, Processing of Microalgal Biomass, Exploitation of Microalgal Products, Microalgae for Biofuels was
added, Wastewater Treatment, and Future Developments and Prospects. Figures 13 were updated showing new cultivation systems. Figure 5 was revised.
Table 1 was revised and updated.

Reference Module in Earth Systems and Environmental Sciences

http://dx.doi.org/10.1016/B978-0-12-409548-9.09373-8

Mass Cultivation of Freshwater Microalgae

exchange represents an artificial system, which is completely different from optically-thin natural phytoplankton populations. It is
desirable to monitor the culture status operatively in order to optimize photosynthetic activity and growth.
This article presents a general overview of freshwater microalgae for biomass production: production strains and basic
techniques and concepts, as well as cultivation systems and, its drawbacks and achievements.

Biological Principles and Technology of Mass Cultivation

Microalgae belong among the fastest-growing photosynthetic organisms since their cell doubling time can be as little as a few
hours. Biomass production by microalgae (oxygenic photosynthetic organisms) is based on the simple scheme shown below,
which determines all the necessary requirements of this biological process:
CO2 + H2 O + nutrients + light energy ! biomass + O2
In biotechnology, generally, the production of biomass requires well-defined conditions. The necessary cultivation requirements for the growth of microalgal mass cultures are light, a suitable temperature and pH, and a sufficient supply of carbon and
nutrients in the growth medium. Since microalgal mass cultures grow in dense suspensions, some kind of turbulent mixing is
necessary to expose cells to light and to allow for an efficient mass transfer.

Light
Light is the most important factor for microalgal growth. The amount of photon energy received by each cell is a combination of
several factors: photon flux density, cell density, length of optical path (thickness of culture layer), and rate of mixing. The ambient
light maxima available for photosynthetic antennae represents an intensity roughly ten times higher (about 2000 mmol photons
m2 s1) than that required to saturate growth. In other words, we have to adjust the optimum culture density for an optimal light
regime and growth otherwise as much as 90% of the photons captured by the photosynthetic antennae may be dissipated as heat.
Therefore, the efficiency of light utilization usually drops from a theoretical value of about 20% (based on photosynthetically active
irradiance, PAR) to 23%, roughly corresponding to an annual biomass yield of about 4060 metric tons per hectare at the latitude
of Central Europe and assuming a mean solar irradiance of 18 MJ m2 days1 for about 210 days of cultivation. Different ways for
reducing the light saturation effect has been proposed to: (i) increase the population density and mixing rate of the cultures; (ii) use
special designs of photobioreactors; and (iii) select suitable strains. One way to reduce the saturation effect of photosynthesis is to
achieve light dilution. This situation can be accomplished by increasing the cross-section of the photobioreactor, i.e. increasing the
illuminated surface of the reactor with respect to the ground area it occupies (see Figure 3(e) and 3(f )). Another important aspect is
that the lightdark cycles of cells facilitated by culture turbulence must be fast, i.e. close to the turnover of the photosynthetic
apparatus, in order to secure a flashing light effect in the range of tens to hundreds of milliseconds (Zarmi et al., 2013). Various
sophisticated photobioreactors for maintaining cells in an ordered lightdark cycle have been designed (Zittelli et al., 2013).

Temperature
After light, temperature is the most important parameter to measure and with which to control the microalgal culture. Some
microalgal strains tolerate a broad range of temperatures between 15 and 40  C (e.g. Chlorella and Arthrospira), while the diatom
Phaeodactylum usually requires a more strict regulation between 20 and 25  C; at 30  C the growth is completely stopped. However,
for the majority of freshwater microalgae, the optimum temperature ranges within 25 and 30  C.

Culture Monitoring and Maintenance

Successful cultivation requires a continuous monitoring of a cultures physicochemical parameters, namely its pH, temperature,
dissolved oxygen concentration, and nutrient status. One monitoring method is to use basic biological examination under the
microscope: in order to detect morphological changes (or cell damage caused by the mixing system) and contamination by other
microalgae, bacteria, fungi and protozoa. The nutrient status can be followed by monitoring the concentration of nitrogen, and
then using this as a measure for adding proportional amounts of other nutrients. In the mass cultivation of microalgae for biomass
utilization, monocultures are usually required. The appearance of contaminants might indicate that the culture has come under
stress. Such contaminants often represent one of the major limitations to large-scale production in microalgal cultures, particularly
with strains that cannot be grown in selected conditions outdoors. For the cultivation of some microalgae (for example,
Haematococcus), the use of a closed system becomes mandatory. In these culture systems it is usually possible to keep the culture
conditions at an optimum and therefore reduce the risk of contamination.
There should be sufficient mixing of the microalgal suspension in order to ensure nutrient diffusion and a homogeneous light
supply to the cells, as well as the need to prevent the accumulation of oxygen in the culture, particularly when they are grown in a
closed system. Indeed, excessive oxygen accumulation in a culture can promote photoinhibition of photosynthesis and bring about

Mass Cultivation of Freshwater Microalgae

a decline in growth. On the other hand, excessive mixing can cause hydrodynamic or sheer stress to the cells, and consequently a
reduction of productivity.
Biophysical and biochemical monitoring methods reflect the general status of the cells photosynthetic apparatus and are thus
often used to adjust the appropriate cultivation conditions: either for the production of biomass or for certain compounds. The
concentration of dissolved oxygen, as measured by an oxygen electrode, is also considered as a reliable and sensitive indicator of
photosynthetic activity in microalgal cultures.
More recently, chlorophyll fluorescence has become one of the most common and useful approaches used for monitoring the
physiological status of microalgal mass cultures (Masojdek et al., 2011). Its non-invasiveness, sensitivity, ease of use, as well as its
prompt provision of results, makes it a convenient and suitable technique in microalgal biotechnology. The ratio Fv/Fm (the
variable to maximum fluorescence yield), the so-called maximum photochemical yield, is considered to be a convenient measure
of the performance of the photochemical processes in photosystem II (the PSII photochemical yield): it relates the utilization of
absorbed light energy to primary production. A decline in the Fv/Fm ratio by about 20% of the value obtained during the morning
can be considered as a reliable warning signal of a certain culture stress that may affect the productivity. The measurement of
relative electron transport rate, ETR (the product of the actual photochemical yield and irradiance intensity), can be used as an
indicator of growth rate.
Culture growth might be estimated as changes in the optical density (OD) at 750 nm, the biomass dry weight, or the number of
cells. The specific growth rate is usually calculated as m(h1) (ln X2  ln X1)/(t2  t1), where X is cell number, or dry weight, at
various successive times. Biomass productivity can be expressed as the areal or volumetric yield per unit time, that is, in
g m2 days1 or in g l1 day1, respectively.
Putting it simply, two basic cultivation regimes are used for the growth of microalgal cultures. In the batch regime, the culture is
inoculated and at a certain point of growth it is harvested. In the continuous regime, the culture is harvested continuously according
to its growth rate and fresh medium is added to replace nutrients. In practice, semicontinuous or semibatch regimes are usually
adopted, that is, where a part of the culture is harvested at regular intervals.

Cultivation Systems
Several alternative cultivation systems and technologies have been developed to grow microalgal mass cultures, using both natural
and artificial light. The choice of a suitable cultivation system and the adjustment of the cultivation regime must be worked out for
each individual productive strain. In every cultivation system, several basic features must be considered: illumination, circulation,
and gas exchange (supply of CO2 and O2 degassing).
Simplifying again, two basic approaches are used in microalgal mass production: the first applies to cultivation in open
reservoirs that are relatively large in area, while the second represents closed vessels photobioreactors or fermenters. In this
article, the term photobioreactor is used for closed or semiclosed systems using natural or artificial illumination. Generally,
production from an open-reservoir culture is cheaper than from a culture in a closed photobioreactor, but the use of the open pond
is limited to a relatively small number of microalgal species. From a commercial point of view, the price of the final product is often
the crucial consideration.

Laboratory Cultivation
The simplest cultivation vessel is an illuminated flask containing the microalgal culture placed on a shaker stock cultures of
microalgae usually being maintained in this way. Glass cylinders or flat flasks kept in a temperature-controlled water bath and
bubbled with a mixture of air with CO2 are commonly used to cultivate microalgae and cyanobacteria in small volumes up to 1 l
(Figure 1).
The transfer to the outdoor culture is scaled up in stepwise fashion, starting with the laboratory culture in a dilution ratio of
approximately 1 to 510. It is advisable not to expose diluted laboratory cultures outdoors to full sunlight during the first few days,
in order to avoid the risk of photoinhibition. However, a minimum biomass concentration corresponding to about 10 g m2
(200 mg chlorophyll m2) is recommended.

Open Outdoor Systems

Natural or artificial ponds, raceways (shallow race-tracks mixed by paddle wheels) and circulation cascades (i.e. inclined-surface
systems) represent open cultivation systems for microalgae with the culture having direct contact with the environment. The
simplest artificial outdoor system is a walled pond, lined with a plastic foil, and mixed by bubbling air (+CO2) through it. An
overview of several open culture systems used for the mass cultivation of microalgae outdoors is presented in Figure 2.
Due to the limited control of cultivation conditions, open systems are suitable for microalgal strains that grow rapidly, or under
very selective conditions. Numerous variations of open ponds are used: according to local requirements, climate conditions, and
materials available (concrete, PVC, fiberglass). Large artificial shallow ponds of 20005000 m2, for example, are used for the
cultivation of the halotolerant microalga Dunaliella in Western Australia to produce b-carotene. Productivity in these ponds is very
low (1 g dry weight m2 day1) due to the lack of mixing and CO2 supply. To improve productivity, tanks, raceways and ponds
are mixed by impellers, rotating arms, paddle wheels, or by a stream of CO2-enriched air supplied into the culture (Figure 2(a)2(c)).

Mass Cultivation of Freshwater Microalgae

The culture depth may vary between 10 and 30 cm. The cultures are usually grown at a biomass concentration ranging between 0.5
and 1 g l1 depending on the culture depth. Outdoor cultures are considered a photo-limited system as they are operated at an
optimum concentration rather than at a maximum growth rate. Open ponds are preferentially used for Spirulina and Chlorella
production in Japan, Thailand, California, Hawaii, Taiwan, India, and China.
In inclined-surface systems, the microalgal suspension flows over sloping planes arranged in cascades, in such a way that the
layer thickness remains below 1 cm and the turbulent flow created by the arrangement prevents self-shading (Figure 2(d)). A high
surface-to-total volume ratio of up to 130 m1 can be operated in these systems and give high volumetric productivities. Due to the
very short optical path, high densities of biomass of between 15 and 35 g l1 can be operated in these units. Such high

Figure 1 Laboratory cultivation using glass cylinders (i.d. 35 mm, volume 0.4 l) placed in a temperature-controlled water bath with adjustable backside
illumination from LED panels (Institute of Microbiology, Academy of Sciences, Trebon, Czech Republic). Mixing of the microalgal suspension is
maintained by bubbling through a mixture of air + 1% CO2.

Figure 2 Examples of open outdoor systems for cultivation of microalgae, which can be scaled up to large production facilities (10,000 litres).
(a) A walled pond lined with a plastic foil and mixed by air (+CO2) bubbling at the Centre for Aquaculture, University of Naples Federico II, Portici,
Italy; (b) a circular pond with a rotating arm (100 l) at the Institute for Ecosystem Study of the CNR (Florence, Italy); (c) a raceway pond with a
paddle-wheel mixer (600 l) at the Faculty of Marine Sciences and Technology, Canakkale Onsekiz Mart University, Turkey), (d) an inclined-surface
system of sloping planes arranged in cascades (90 m2; 6001000 l) at the Institute of Microbiology, Trebon, Czech Republic).

Mass Cultivation of Freshwater Microalgae

productivities as over 40 g dry matter m2 day1 can be achieved in cascade cultivation units, even in temperate climate zones. Yet,
up till now, the system has not been tried fully scaled up, due to its higher construction costs compared to traditional ponds;
however, the system is transportable with a long working life.
Although open systems cost relatively less to build and operate, and are more durable with a larger production capacity,
compared to more sophisticated photobioreactors, open systems have intrinsic disadvantages, such as: difficulty in managing a
suitable culture temperature and light availability on a per cell basis; a massive water loss due to evaporation; a susceptibility to
microbial contamination; and a low cell concentration and biomass productivity.

Closed and Semiclosed Outdoor Photobioreactors

Closed photobioreactors are flexible systems that can be optimized according to the biological and physiological characteristics of
the microalgal species involved. An overview of several closed or semiclosed culture systems used for mass cultivation of microalgae
outdoors is presented in Figure 3. The simplest cultivation system is an illuminated bag containing the microalgal suspension,
mixed by a stream of CO2-enriched air (Figure 3(a)). Compared to open systems, photobioreactors have a number of advantages:
reproducible cultivation conditions with regard to environmental influences; reduced risk of contamination; low CO2 losses; and
smaller area requirements. On the downside, closed systems are: more difficult to clean; the tube material might partially decrease
sunlight penetration; and the system must be cooled and degassed effectively since any excessive oxygen produced by the growing
cultures can reduce growth. Furthermore, the cost of construction is about one order of magnitude higher than that of open ponds
(about 100 US$ m2).
A schematic diagram of a closed photobioreactor is illustrated in Figure 4. Either Plexiglas, acrylic and glass tubes, flat and
alveolar panels, or flexible plastic coils can act as a photostage through which the microalgal suspension is being continuously
circulated. Here, a much greater biomass density can be maintained than in open ponds. Outdoor photobioreactors for commercial production are usually designed as modules.
Generally, the tubes are positioned horizontally or vertically, arranged as a serpentine loop or as manifold rows. The culture
suspension is circulated by a pump or more preferably by air-lifting (injecting a stream of compressed air in an upward-pointing
tube). Peristaltic and membrane pumps are physically more friendly to cells than centrifugal pumps, which can cause higher sheer
stress. Cooling is maintained by submerging the tubes in a pool of water, by heat exchangers, or by spraying water onto the
photobioreactor surface. A further improvement is represented by the two-plane horizontal tubular photobioreactor built in
Florence, Italy, which led to a high Spirulina productivity of 30 g dry weight m2day1 (Figure 3(b)). The biggest production plant
to date has been built in a greenhouse in Klotze and in Ritscherhausen (Germany), with a volume of 700 m3 (20 modules of
35 m3). Horizontal glass tubes are arranged in a vertical fence-like system in order to utilize diffuse light (Figure 3(c)). Recently,
IGV Biotech supplies glass tube photobioreactors of up to 3000 l mounted in mobile containers (e.g. used in a system for
sustainable aquaculture of microalgae-fish).
Helical tubular systems, commonly called biocoils, seem to be another alternative for microalgae cultivation (see diagram in
Figure 4). It consists of coiled, flexible, transparent tubes (36 cm diam.) around an open cylindrical frame, realized by the company
Addavita Ltd. in the UK. In Australia, 100-l laboratory photobioreactors have been scaled up to 1000-l outdoor pilot plants.
Flat-panel photobioreactors with a short light path, with a horizontal, inclined, or vertical orientation, have a high surface-tovolume ratio. These photobioreactors are made from Plexiglas or polycarbonate alveolar sheets, 24 cm thick, with flexible,
polyethylene bags enclosed in a rigid framework (Figure 3(d)).
A similar principle of cultivation has been used in flat photobioreactors developed as transparent flat-panels connected in series,
vertically-arranged 20 cm apart (Figure 3(f )). Compared with tubular systems, flat-panel photobioreactors have one serious
disadvantage: a fouling up of the channels due to reduced turbulence in their narrow, rectangular shape channels.
Vertical-column photobioreactors are relatively simple systems in which mixing is achieved by air + CO2 bubbling up from the
bottom. A variation of this column system are annular photobioreactors, which consist of two glass or Plexiglas cylinders of different
diameters placed one inside the other to form a culture chamber some 510 cm thick and 50200 l in volume (Figure 3(e)).
Illumination can be provided by either natural or artificial light. In column photobioreactors, sensitive strains with fragile cells or
filaments can be grown as the culture mixing is very gentle (e.g. Nostoc, Isochrysis, Navicula, Skeletoma, etc.). Despite the higher yields
attainable with photobioreactors, their high construction and maintenance costs still make them uncompetitive for the industrial
production of microalgal biomass. Their use can be foreseen for the production of high-value bioactive substances, which require
the adoption of sterile conditions.

Heterotrophic Fermenters
Microalgae are generally thought of as phototrophic microorganisms; however, some strains grow on sugar in the dark (heterotrophic production). Heterotrophic fermenters for the cultivation of microalgae were tested during the 1970s, as some microalgal
strains (e.g. Chlorella, Chlamydomonas, Phaeodactylum, and Haematococcus) can grow heterotrophically. By the mid-1990s, heterotrophic production technology had gained in industrial importance for Chlorella biomass production in Japan and remains so
today. Commercial fermenters come in a wide range of sizes: from 10 to 100 000 l. Photobioreactors and fermenters have many
features in common: pH and temperature control, harvesting, mixing, degassing, etc. Compared to photobioreactors, the

Mass Cultivation of Freshwater Microalgae

Figure 3 Examples of closed or semiclosed photobioreactors for cultivation of microalgae, which can be scaled up to large production facilities. (a)
Hanging plastic bags mixed by air (+CO2) bubbling (Faculty of Marine Sciences and Technology, Canakkale Onsekiz Mart University, Turkey); (b) a
two-plane, horizontal tubular photobioreactor (Institute for Ecosystem Study of the CNR, Florence, Italy); (c) vertically stacked tubular photobioreactor
mounted in a greenhouse developed by IGV GmbH (Salata GmbH, Germany); (d) a vertical flat-panel photobioreactor Green Wall Panel (Institute for
Ecosystem Study of the CNR, Florence, Italy); (e) A 100-liter annular column photobioreactor consisting of two glass cylinders placed one inside the
other to form the culture chamber; the LED light source is mounted in the internal cylinder and can be placed outdoors to combine natural and artificial
light (Institute of Microbiology, Trebon, Czech Republic); (f) An innovative flat-plate photobioreactor Hanging Gardens developed by ecoduna GmbH
(Bruck a/L, Austria) consisting of 12 closely spaced parallel panels (0.03  2  6 m) placed on a movable frame that allows to track the sun movements.
The panels are internally partitioned by baffles to allow culture circulation as air and CO2 are injected from the bottom to generate a gas-lift effect.

significant differences of fermenters are their energy source, oxygen supply, and sterility, as well as some advantages such as high
biomass yield, lack of a light requirement, and ease of monoculture control. When grown heterotrophically, microalgal cultures
utilize some organic compound (e.g. glucose or acetate) as both a carbon and energy source for growth. The crucial requirement is
that the microalgal cultures must be axenic (i.e. free from other microorganisms) to avoid the growth of contaminants. It is
absolutely essential that the fermenter and culture medium are sterilized before inoculation (for example, by steam). In fermenters,
adequate mixing is achieved with impellers and a stream of compressed air as a source of oxygen for the catabolic processes.

Mass Cultivation of Freshwater Microalgae

Figure 4 Schematic diagram of a photobioreactor. It consists of a photostage loop, heat exchanger, degasser, circulation pump, CO2 supply, and
sensors (e.g., pH and oxygen electrodes, and thermometer). Created by PalSek.

A biomass density up to 100 g l1 can be obtained in fermenters with a volumetric productivity higher than 10 g l1 day1.
Recently, the microalga Crypthecodinium cohnii has been used industrially in the production of docosahexaenoic acid (DHA; C22:6,
o-3) via heterotrophic fermentation. It is a two-stage fed-batch process a cell growth phase is carried out under a full nutrient
supply, while during the lipid production phase the nitrogen is limited.

Biotechnologically Important Strains of Microalgae

About 100 000 strains of microalgae have been isolated from natural habitats and are kept in numerous culture collections around
the world (e.g. UTEX, Austin, USA; IAM, University of Tokyo, Japan; SAG, Culture Collection of Algae at the Gottingen University;
CCALA, Trebon, Czech Republic; ISE-CNR, Italy). However, to date, relatively few microalgal strains, mostly of aquatic origin, have
been cultivated in large-scale production systems of hundreds to thousands of liters. Basically, these strains belong to two groups of
unicellular or filamentous photosynthetic microorganisms prokaryotic cyanobacteria and eukaryotic algae. Many advances have
been made in their mass production, processing and application in the last 40 years.

Arthrospira (Spirulina)
Arthrospira platensis is a planktonic filamentous cyanobacterium composed of individual cells (about 8 mm diam.), which grows in
subtropical, alkaline lakes with a temperature optimum of about 35  C. In productive cultures, Arthrospira is cultivated in shallow,
mixed ponds or semi-closed, tubular photobioreactors. The growth medium contains inorganic salts with a high concentration of
bicarbonate, keeping the pH value between 9 and 10. This cyanobacterium is the most cultivated photosynthetic prokaryote, since
its biomass is widely used as a health food, feed supplement, and as a source of fine chemicals. The total annual production of
biomass is estimated at about 12 000 metric tons, more than 50% of which is in China and the Asia-Pacific region. It contains
proteins (60%), carbohydrate (15%), lipids, phycobiliproteins, carotenoids, vitamins, and minerals.

Chlorella
Chlorella (green algae; Chlorophyta) is a cosmopolitan genus with small globular cells (about 2-10 mm diam.) living in both aquatic
and terrestrial habitats. It includes strains with a high temperature tolerance, since some strains can grow between 15 and 40  C.
Due to its simple cell cycle, high growth rate, and having photosynthetic and metabolic pathways similar to higher plants, Chlorella
has long been used as a model microorganism for studying the photosynthetic apparatus and carbon assimilation. Chlorella strains
grow phototrophically in an inorganic medium as well as in mixotrophic and heterotrophic conditions (e.g. with the addition of
acetic acid and glucose). At present, phototrophic production of Chlorella is carried out in open ponds, semiclosed tubular
photobioreactors, or inclined cascades, since its high growth rate prevents contamination by other microalgae (e.g. in Germany,
Japan, China, Czech Republic, and several other Asian countries) with a total annual production of about 5000 metric tons. The

Mass Cultivation of Freshwater Microalgae

processing of Chlorella cells requires both an effective and efficient harvesting (flocculation, flotation, centrifugation, etc.) and a
mechanical disintegration of the cellulose cell wall.
Chlorella is the most cultivated eukaryotic alga since it is widely used as a health food and feed supplement, as well as in the
pharmaceutical and cosmetics industry. It contains proteins (up to 60% of dry weight), polysaccharides (1015%), lipids (12
15%), unsaturated fatty acids, and carotenoids (predominantly lutein), as well as some immunostimulators, vitamins, and
minerals. It is important to note that Chlorella also possesses the ability to synthesize larger amounts of storage compounds
(polysaccharides or neutral lipids) when under stress conditions, e.g. under high irradiance with nutrient deficiency, making it a
promising source for biofuel production.

Dunaliella
The green halophilic microalga Dunaliella salina (Chlorophyta) and similar hypersaline strains have biflagellated, pear-shaped cells.
Dunaliella is the main natural source of b-carotene in high amounts, it being up to 16% of dry matter. Their cells lack a rigid cell
wall, having instead a thin elastic plasma membrane. This microalga is a natural source of carotenoids for human use as well as for
animal feed (shrimps). The high content of b-carotene makes Dunaliella attractive to biotechnologists for large-scale production in
high-salinity, shallow, open ponds under high solar radiation (>30  C). In the cells, b-carotene is usually accompanied by other
carotenoids (astaxanthin and canthaxanthin), which are all accumulated in oily globules in the chloroplast. Natural b-carotene is
marketed in various forms: b-carotene extracts, Dunaliella powder for human use, and dried Dunaliella for feed coloration.
Currently, there are large Dunaliella production plants in Australia and Israel.

Haematococcus
Haematococcus pluvialis (Chlorophyta) is a freshwater, unicellular green microalga with a rather complex life cycle. Among various
natural sources, Haematococcus is an exclusive producer of astaxanthin (pink carotenoid). The biosynthesis of astaxanthin is usually
accompanied by the transformation of ovoid green vegetative cells into red cysts under stress conditions (nutrient deficiency,
salinity, and high temperatures, in combination with high irradiance), due to the increased carotenoid deposition. When the
condition becomes favorable for growth, the cysts germinate releasing a large number of new motile cells.
The Haematococcus strains grow slowly and is commonly carried out in open raceway ponds or closed photobioreactors at
around 2528  C, and are prone to contamination by other microorganisms (microalgae, fungal parasites, and zooplankton
predators). Therefore, a two-stage process is usually employed for biomass production. Green vegetative cells are usually produced
in closed photobioreactors under an optimal light intensity and nutrient-replete medium. Then, at maximum cell density, the
culture is pushed towards a red stage aplanospores by exposure to high irradiance in open systems under nutrient stress in
order to induce astaxanthin synthesis (up to 5% of dry weight) within 35 days. This pigment is important for human nutrition as
an anti-oxidant (protection agent against free-radical-induced diseases) and a natural colorant for the aquaculture of salmonoid
fish, shrimp, lobster, and crayfish. However, today, the production of astaxanthin is still restricted to that of a few hundred kilos,
mainly addressed to the health food market. The actual production costs are still too high to compete with synthetic equivalents.
Although the commercial market is dominated by the synthetic product, there are concerns about its safety for human consumption, which makes natural astaxanthin a preferred choice.

Microalgae for Aquaculture

Microalgae cultivation is a part of the global aquaculture industry as a number of species are used in aquaculture, for example:
Nannochloropsis (Eustigmatophyceae); Chlorella, Dunaliella, and Haematococcus (Chlorophyceae); Isochrysis and Pavlova
(Prymnesiophyceae); Tetraselmis (Prasinophyceae); and Phaeodactylum, Skeletoma and Navicula (Bacillariophyceae). The aquaculture potential for microalgae cultivated in mass cultures is mostly to enhance the nutritional content (proteins, carotenoids, fatty
acids, etc.), which positively affects the health and physical condition of the produced organisms. There exist a number of different
modes in which microalgae are utilized for the larvae of molluscs, crustaceans, fish hatchings, or rotifers in aquaculture that are all
sources of biomass directly as unprocessed cells (live feed), to build up the respective food chain, or in the form of processed
biomass to be added to the diet. Aquaculturists often manage microalgae cultivation in-house for dietary purposes, using various
types of closed photobioreactors or outdoor open systems. Apart from the nutritional purposes, the other important impact of
microalgae is the improvement of the environment in fish, crustacean and mollusc aquaculture as concerns the production of
oxygen, consumption of CO2, waste nutrients and bactericidal activity. For the larval and juvenile stages of numerous freshwater
and marine species, the introduction of phytoplankton to rearing ponds, the so-called green-water technique, has produced much
better results in terms of survival and growth than the more traditional clear-water techniques.

Processing of Microalgal Biomass

A schematic diagram of microalgal biomass production and processing is shown in Figure 5. After cultivation, the harvesting of
single cells or filaments is done by centrifugation, filtration, flotation or flocculation the selection of the actual harvesting method

Mass Cultivation of Freshwater Microalgae

Figure 5 A schematic diagram of microalgal biomass production and processing. Microalgae are grown in a cultivation unit in the aqueous mineral
medium under illumination, and nutrient and CO2 supply. The biomass is separated from the medium and processed (disintegrated and dried). The
biomass can be used as a food or feed supplement, health food, or as a source of bioactive substances for pharmacology and cosmetics.

depending on the dimensions of the organism. Some filamentous and large-cell strains can be separated by filtration using
vibrating screens. Flocculation is the collection of cells into aggregates by the addition of multivalent cations, metal salts or
polymers (for example, polyaluminum chloride). This method is frequently used to remove natural water blooms. Bioflocculation
using chitosan (a non-toxic polymer of acetylglucosamin), autoflocculation by pH change, or co-flocculation of mixed cultures of
particular microalgae, have also been successful. Flotation is an air-supported separation in which microalgal cells are attached to
air bubbles to form flocks that float up to the surface for harvesting. Membrane filtration is also used for microalgae harvesting, but
a serious obstacle is membrane fouling. Recently, magnetic separation (the capture of microalgal cells by magnetic particles of
Fe3O4) has been used for the removal of microalgae from thin cultures, but the practical application of this technique is still being
limited by the difficulties in the production of particles. A good harvesting and dewatering process may produce microalgal slurry
of 2030% biomass content.
The disruption of microalgal cells is an important operation in biomass processing, since some cells have a tough cell wall. This
is done mechanically in homogenizers, bead beaters, and ultrasonic homogenizers, or by chemical methods.
Dehydration of the microalgal slurry is achieved by solar drying, spray drying, or lyophilisation. The latter freeze drying is the
gentlest method since it is based on the sublimation of water from the frozen biomass under vacuum. In spray driers, the
concentrated suspension of microalgal cells is sprayed into hot air in a closed chamber and the dried biomass collected. In this
process, rigid cell walls can also be ruptured. Spray drying is the most common method on an industrial scale for nutritional use.
The harvesting and drying process may contribute about 25% of the total costs of microalgal biomass production.

Exploitation of Microalgal Products

As phylogenetically the oldest organisms, microalgae have adapted to a wide range of extreme habitats. This fact has resulted in the
development of numerous protective systems against various stressors high irradiance in combination with salinity, temperature
extremes, desiccation, nutrition deficiency, etc. To these ends, microalgae produce various storage or protective substances such as
polyunsaturated fatty acids (PUFA), lipids, antioxidants, or immunologically effective, virostatic, and cytostatic compounds.
Therefore, they are cultivated commercially for biomass as food and feed additives, and as a source of bioactive and novel
compounds for pharmacology, cosmetics and the chemical industry, or, on a small scale, for research or diagnostic products.
Opportunities for novel pathway discoveries and genetic manipulation have to be considered in a biosynthetic perspective.
The biomass as a powder, concentrated suspension, or in some other applied form, is the primary product. In most countries,
microalgae can be legally marketed as long as the product is labelled accurately and contains no contamination, or harmful
substances. It should be an unwritten agreement among the producers of microalgae and derived products that these shall meet all

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Mass Cultivation of Freshwater Microalgae

Table 1

The current industrial-scale biotechnology applications of the most exploited microalgal strains

Product and application

Status

Microalga

Health food, food and feed supplements

b-Carotene
Astaxanthin
Live food and feed supplements in aquaculture

Established
Established
Established
Established

PUFAs

Established

Xanthophylls (lutein, zeaxanthin)

Polysaccharides
Oils

Developing
Developing
Developing

Arthrospira (Spirulina), Chlorella

Dunaliella
Haematococcus
Isochrysis, Nannochloropsis, Chaetoceros,
Pavlova, Tetraselmis, Thalassiosira, Phaeodactylum, Skeletonema, etc.
Crypthecodinium, Phaeodactylum (Nannochloropsis,
Nitzschia, Porphyridium)
Scenedesmus, Chlorella
Porphyridium
Botryococcus

the relevant food quality and safety standards, and also follow good manufacture practice (GMP) guidelines that cover all aspects of
food processing.
The current industrial-scale biotechnology applications of the most exploited microalgae are summarized in Table 1.

Nutrition
For centuries, microalgae, mostly Arthrospira, have been used as a food supplement by native tribes in Mexico, Africa and Southeast
Asia. In the past five decades, there have been numerous attempts by researchers and companies to commercialize microalgal
production, primarily as food and feed supplements, due to its potential to enhance the nutritional value of conventional food and
as probiotics (life-enhancing agents). The nutritional properties of microalgae have been obtained from a broad spectrum of
studies of both humans and animals. It contains proteins, essential aminoacids, carbohydrates, lipids including PUFAs, pigments,
antioxidants, nucleic acids, raw fiber, vitamins, minerals, and more. Today, microalgal biomass, mostly of Chlorella and Spirulina, is
marketed as health food and as a protein source in the form of tablets, capsules, and liquids. The annual microalgal market is in
thousands of tons. Microalgal biomass is incorporated into pasta, snacks, drinks, and beverages as a nutritious supplement or
colorant. Plant proteins are a source of some essential amino acids that humans and animals cannot biosynthesize (e.g. methionine, lysine, and tryptophan).
Various unsaturated fatty acids in microalgal biomass are important as dietary supplements to prevent various diseases
(e.g. high plasma cholesterol, cardiovascular diseases, hypertension, arteriosclerosis, arthritis, etc.) and to boost the immune
system. Microalgal biomass also contains all the important vitamins, especially the B1, B2, B12, K, E and C vitamins and nicotinic
acid. The variety of carotenoids is greater than that in higher plants b-carotene, astaxanthin, canthaxanthin, lutein, violaxanthin,
zeaxanthin, neoxanthin, amongst others. Among them, the oxygenated xanthophylls, astaxanthin and canthaxanthin, are massively used both as colorants and antioxidants in aquacultures (fish and Crustacea).
Microalgal biomass is also widely used as a feed supplement (13%) for poultry, ruminants, pigs, ornamental fish, and birds, to
improve the quality of their products, vitality, health resistance, and color of hair and skin.

High-value and Bioactive Compounds

Microalgae are an ideal platform for the large-scale production of high-value products because they represent fast-growing, solarpowered biofactories with low nutrient requirements!
Only plants synthesize unsaturated fatty acids (linoleic, linolenic, arachidonic, eicosapentaenoic, and docosahexaenoid acid).
Therefore, microalgae can supply whole food chains with these vital components. Microalgal PUFAs have a very promising
biotechnological market both for human food and animal feed.
For lipid-based cosmetics, such as creams or lotions, ethanolic or supercritical CO2 extracts of microalgal biomass are also
gaining commercial importance, because they provide both nourishing and protective effects to human skin.
Microalgal polysaccharides are other pharmacologically-important compounds. For example, certain highly sulfated polysaccharides trigger either a cellular or humoral stimulation of the human immune system. Effective polysaccharide fractions
have also been found mainly in cyanobacteria; however, compounds from green and red microalgae are also efficient.
Due to their phototrophic life, microalgae experience high oxygen and radical stresses. Their protective mechanisms, based on
highly effective oxygen radical scavengers, prevent the accumulation of free radicals and reactive oxygen species (e.g. superoxide
anions, hydroxyl radicals, or singlet oxygen), thus avoiding cell damage. For example, the antioxidative potential of Spirulina can
increase 2.3-fold during oxidative stress. Of particular note is the high content in lipophilic scavengers, such as carotenoids,
especially b-carotene, a-tocopherol, lutein and astaxanthin; minerals and other trace elements with an antioxidative effect, such as
zinc and selenium; and enzymatic scavengers, such as catalase, superoxide dismutase, and peroxidase, and the vitamins C and E.

Mass Cultivation of Freshwater Microalgae

11

The most impressive demonstration of the ability of microalgae to produce highly effective bioactive compounds are microalgal
toxins, which in blooms become dangerous to animals and humans, especially if it occurs in drinking-water reservoirs (found
especially in Microcystis, Anabaena, and Aphanizomenon).
The extensive screening of microalgae for new substances with biological activities bioprospecting undertaken in many
laboratories and companies, has revealed secondary metabolites with biological activities in extracts: antiviral, immunomodulatory, cytotoxic important in anticancer drugs, antimicrobial for finding new antibiotics, and antifungal.
Because phototrophic microalgae can be cultivated under strictly controlled conditions, they are the ideal choice to incorporate
stable isotopes from inorganic C-, H-, and N-sources. Various biochemicals labelled by stable isotopes are used for scientific
purposes (molecular structure or physiological investigations), as well as for clinical purposes (gastrointestinal or breath
diagnostic tests).

Microalgae for Biofuels

Microalgae have a significant potential, compared to other biomass feedstocks, to supplement current transportation fossil fuels.
Some species, such as Nannochloropsis, or Chlorella can accumulate as much as 60% of storage compounds (lipids or polysaccharides) under nutrient limitation. There are several advantages offered by microalgae over higher plants as a source of biodiesel or
bioethanol, for example: (1) the yield of storage compounds per unit of area for microalgae can greatly exceed that of traditional
crops; (2) microalgae can be cultivated in areas unsuitable for conventional agriculture; (3) they can be cultured in controlled
aquaculture; (4) microalgae grow either in seawater or in brackish water which is unusable for normal agriculture; and (5) microalgae allow the recovery of P and N from wastewater, and the utilization of waste CO2 (e.g. from flue gases). On the other hand, the
cultivation of microalgae is a more expensive process than that of conventional crops since it requires energy for culture mixing,
harvesting and biomass processing. The minimum current cost of microalgal biomass production is about 5 US$ per kg, but it still
exceeds by one to two orders of magnitude the rate required for economic biofuel production. The cost reduction is based on
suitable and novel strain selection, low-cost photobioreactors, and lower energy inputs. The current technology is rather unlikely to
produce a positive energy and carbon balance for microalgal biofuels as an alternative energy source. However, finding additional
value in biomass residues may greatly enhance the viability of biofuel technology. Nutrient supplies through wastewater treatment,
combined with the use of residual biomass for methane production, could potentially support the likelihood of producing biofuels
that could compete with conventional fuels. Efforts to produce biofuels from microalgae are worthy of study, but one must be
aware that this technology may only become economically viable within 1015 years.
Microalgal cultures have been used in high-rate oxidation ponds, aimed at the exploitation of biomass for alternative biofuel
feedstocks. Microalgae-remediation systems absorb nutrients and produce oxygen by photosynthesis which enhances the growth of
bacteria used in degrading organic matter.

Ecological Application of Microalgal Mass Cultures

Environmental biotechnology is a rapidly emerging area which is dedicated to the research and application of biological processes
for the remediation of contaminated environments (water, soil, air). Ecologically-designed human communities might incorporate
microfarms: with controlled cultivation in a greenhouse or bioreactor. On a relatively small land area, a community could meet a
significant portion of its protein and vitamin requirements from microalgae, thus freeing up present agricultural cropland for
community recreation or for reforestation. In the 1990s, a very early attempt was made to use a CO2-containing chimney-stack gas
produced in a lime kiln as a source of carbon dioxide for a plastic-plate and glass-tube microalgal photobioreactor of 10 000 l in
Elbingerode (Germany) (well-mixed microalgal culture of high biomass density (>0.5 g l1) with sufficient nutrition and gas
exchange).
For example, a scheme for sustainable aquaculture, through the integration of algaculture, has been recently worked out (Neori,
2011). The technologies that will be needed, though simple in principle, will require a thorough understanding of the biology of
each organism and of the nutrient recycling processes involved. These integrated aquaculture systems efficiently extract dissolved
nutrients, excess CO2, and recharge the system with dissolved oxygen making the treated water either suitable for reuse in
aquaculture or for clean discharge to rivers.
Most advanced farms designed to produce high-quality microalgal biomass necessarily have higher production costs. To lower
costs, the ecological farms of the future need to integrate their sources of nutrients and energy, and produce a variety of end
products, from valuable extracts to inexpensive protein.

Wastewater Treatment
Microalgal cultures, sometimes in combination with other microorganisms, are utilized to treat municipal, agricultural, food and
industrial wastes, as well as aqua- and mariculture effluents for the reduction of environmental loads. Microalgae are suitable for
biofiltration because they grow fast and can be easily cultured under favorable climatic conditions. The key substances contributing
to water eutrophy, for example, nitrate and phosphate, as well as important industrial and agricultural waste gases (e.g. ammonia
and carbon dioxide) are the main nutrients for microalgae.

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Mass Cultivation of Freshwater Microalgae

In contrast to domestic wastewaters, which are basically treated by similar methods all over the world, each industrial
wastewater requires case-specific technology. In addition, many industrial effluents are toxic to microorganisms.
A number of applications have been developed in the following fields:

Development of plants for the disposal of inorganic loads, especially nitrate and phosphate from aquaculture recirculation, by
microalgae
Disposal of contaminants from agricultural waste-water;
Heavy-metal biosorption by microalgal cultures;
Utilization of carbon dioxide from industrial exhaust gas.

Heavy metals (metals and metalloids with a mass density > 5 g cm3) are stable and persistent environmental contaminants since
they resist most forms of degradation. Elevated concentrations of copper, cadmium, lead, mercury, chromium, zinc and nickel are
toxic to most microorganisms. Microalgae have been used to remove and/or detoxify heavy metals in aquatic environments, since
they have a remarkable ability to take up and accumulate heavy metals and have been used for the bioremediation of metalpolluted sites.
Various types of waste-water treatment ponds are widely used all over the world. The symbiotic activity of microalgae and
bacteria is a common concept. However, removal rates of nitrogen and phosphorus are relatively poor and the imperfect removal of
abundant micropollutants, such as pharmaceuticals, medicines, and hormones, makes it impossible to discharge the resulting
effluents to streams; some advanced and integrated processes have to be applied beforehand.

Future Developments and Prospects

The one approach that does not involve genetic manipulation of the producer organism should include: (1) a deepening of our
biological knowledge of existing microalgal species and their metabolites; (2) the discovery of novel producers and potential
products; (3) the optimization of growth conditions; and (4) the optimization of production processes and technology. The
technology of advanced screening that involves metabolomic databases, as well as ecological field surveys, to help identify and
even design novel compounds with various biological activities will support the search for new strains and products. The stages
needed for optimization rely on the basic biology of microalgae being coupled to bioengineering and will require intensive
research.
Genetic engineering represents the other approach to microalgal biotechnology. Cyanobacteria are fairly simple to genetically
alter in comparison to eukaryotic microalgae. There exist several barriers that challenge exogenous DNA before it can be integrated
into a microalgal genome. These can include the cell wall, and several additional membranes, depending on the target organelle
and the species being transformed.
Microalgae have been a promising platform for high-value recombinant proteins because these microorganisms can be grown
on a large scale. Recombinant proteins such as vaccines, therapeutic antibodies, and industrial enzymes, can be produced in
microalgae at relatively low cost. Some highly-valuable recombinant proteins have been produced in the chloroplasts of Chlamydomonas reinhardtii, which was the microalgal strain where many of the transformation techniques were first developed and then
applied to other microalgal groups.
The last two decades have brought significant advances in microalgal molecular biology. Although most progress has been
achieved in a few model systems, we are still not in a position to standardize our molecular and metabolic manipulation of
commercially-relevant species. A much better understanding of the mechanisms that control the regulation of gene expression in
microalgae is still required.

Acknowledgements
The authors thank Dr Magda Sergejevova, Mr Jose R. Malapascua and Mr Pavel Soucek for discussion and technical assistance and
Mr Steve Ridgill for language corrections. The Ministry of Education, Youth and Sports and the Czech Academy of Sciences
supported this work through the project Algatech CZ.1.05/2.1.00/03.0110 and Algain CZ.1.07/2.3.00/30.0059.

References
Masojdek J, Vonshak A, and Torzillo G (2011) Chlorophyll fluorescence applications in microalgal mass cultures. In: Suggett DJ, Prasil O, and Borowitzka MA (eds.) Chlorophyll a
fluorescence in aquatic sciences: methods and applications, pp. 277292. Dordrecht: Springer.
Neori A (2011) Green water microalgae: the leading sector in world aquaculture. Journal of Applied Phycology 23: 143149.
Zarmi Y, Bel G, and Aflalo C (2013) Theoretical anaysis of culture growth in flat-plate photobioreactors the essential role of timescales. In: Richmond A and Hu Q (eds.) Handbook of
microalgal culture: applied phycology and biotechnology, 2nd edn., pp. 205224. Oxford: Wiley Blackwell.
Zittelli GC, Biondi N, Rodolfi L, and Tredici MR (2013) Photobioreactors for mass production of miocroalgae. In: Richmond A and Hu Q (eds.) Handbook of microalgal culture: applied
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Further Reading
Andersen RA (2005) Algal culturing techniques. Amsterdam: Elsevier Academic Press.
Barsanti L and Gualtieri P (2006) Algae: anatomy, biochemistry, and biotechnology. Boca Raton, FL: CRC Press.
Gordon R and Seckbach J (2012) The science of algal fuels. Dordrecht: Springer.
Falkowski PC and Raven JA (2007) Aquatic photosynthesis, 2nd edn. Princetown: Princetown University Press.
Graham LE and Wilcox LW (2000) Algae. Upper Saddle River, NJ: Prentice-Hall.
Larkum AWD, Douglas SE, and Raven JA (2003) Photosynthesis in algae. Dordrecht: Kluwer Academic Publishers.
Pulz O, Scheibenboden K, and Gross W (2001) Biotechnology with cyanobacteria and microalgae. In: Reed G (ed.) Special processes: biotechnology, 2nd edn., vol. 10pp. 107136.
Weinheim: Wiley-VCH.
Rai LC and Gaur JP (2001) Algal adaptation to environmental stresses: physiological, biochemical and molecular mechanisms. Berlin: Springer.
Richmond A and Hu Q (eds.) (2013) Handbook of microalgal culture: applied phycology and biotechnology, 2nd edn. Oxford: Wiley Blackwell.
Van den Hoek C, Mann DC, and Jahns HM (1995) Algae: an introduction to phycology. Cambridge: Cambridge University Press.