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The development of microalgal biotechnology in the Czech Republic

Article  in  Journal of Industrial Microbiology · December 2010

DOI: 10.1007/s10295-010-0802-x · Source: PubMed

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J Ind Microbiol Biotechnol (2010) 37:1307–1317
DOI 10.1007/s10295-010-0802-x

ORIGINAL PAPER

The development of microalgal biotechnology

in the Czech Republic
Jiřı́ Masojı́dek • Ondřej Prášil

Received: 22 June 2010 / Accepted: 30 July 2010

Ó Society for Industrial Microbiology 2010

Abstract Microscopic algae and cyanobacteria are Introduction

excellent sources of numerous compounds, from raw bio-
mass rich in proteins, oils, and antioxidants to valuable Microalgae1 represent a diverse group of microorganisms
secondary metabolites with potential medical use. In the of tremendous ecological importance, the spread of which
former Czechoslovakia, microalgal biotechnology devel- is enormous since they inhabit all major ecosystems—from
oped rapidly in the 1960s with the main aim of providing cold, Arctic regions, through extremely alkaline or saline
industrial, high-yield sources of algal biomass. Unique habitats, to hot springs and arid soils. Prokaryotic cyano-
cultivation techniques that are still in use were successfully bacteria, in particular, represent the oldest group of pho-
developed and tested. Gradually, the focus changed from tosynthetic organisms which started the formation of the
bulk production to more sophisticated use of microalgae, Earth’s oxygenic atmosphere more than 2.5 billion years
including production of bioactive compounds. Along the ago. Microalgae are also responsible for almost half of
way, better understanding of the physiology and cell global primary biomass production and form the basis of
biology of productive microalgal strains was achieved. the food chain in aquatic environments. Furthermore, they
Currently, microalgae are in the focus again, mostly as represent one of the most efficient converters of solar
possible sources of bioactive compounds and next-gener- energy to biomass.
ation biofuels for the 21st century. Algal biotechnology has been closely related to the use
of macroalgae (e.g., Porphyra), which dates back to the
Keywords Green algae
Chlorella
Biotechnology
first millennium. In Asia, these species have been culti-
Biomass productivity
Mass culture vated since the Middle Ages, and today this technology
represents an industry with an annual turnover of billions
of US dollars. For example, the first reports about agar
Abbreviations
production from Gracilaria date back to the 17th century in
DW Dry weight
Japan, and brown algae were already processed for iodine
PSII Photosystem II
and soda in the 18th century [62].
In nature, water blooms of microalgae can develop in
eutrophic reservoirs where phytoplankton populations are
occasionally mixed by wind or flux. Even under these
This article was compiled and used as knowledge source during optimal situations, biomass concentration is much below
preparation of the project Centre for Algal Biotechnologies Třeboň
(Algatech) CZ.1.05/2.1.00/03.0110 applied to the Ministry of
1 g dry matter per liter. Dense, well-mixed mass cultures of
Education, Youth and Sports. microalgae ([0.5 g biomass per liter) represent artificial

J. Masojı́dek (&)
O. Prášil

1
Department of Autotrophic Microorganisms, In applied phycology, ‘‘algae’’ refers to macroalgae well as
Institute of Microbiology, Academy of Sciences, microalgae. The term ‘‘microalgae’’ is usually used in its broadest
Opatovický mlýn, 37981 Třeboň, Czech Republic sense to mean both prokaryotic cyanobacteria and eukaryotic algae—
e-mail: [email protected] unicellular or filamentous photosynthetic microorganisms.

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1308 J Ind Microbiol Biotechnol (2010) 37:1307–1317

Table 1 Biotechnological
Microalga Status Product and application
applications of the most
exploited microalgae Arthrospira (Spirulina) Established Health food, food and feed supplement
Chlorella Established Health food, food and feed supplement
Dunaliella Established b-Carotene
Haematococcus Established Astaxanthin
Nannochloropsis, Isochrysis, Pavlova, Established Source of lipids, fatty acids, and
Tetraselmis, Monodus polyunsaturated fatty acids (PUFAs);
aquaculture feed, biofuels
Microalgal biomass in general Rising Biofuels—biodiesel, bio-oil, bioethanol, etc

systems with sufficient nutrition and gas exchange, which between open and closed photobioreactors, and conse-
are completely different from optically thin natural phy- quently the growth physiology of the microalgae is dif-
toplankton populations with low biomass density, often ferent between the two systems. Several factors governing
limited by nutrient and carbon supply. growth can, within certain boundaries, be manipulated.
Natural sources of microalgae readily available for Crucial variables are the optical depth, turbulence, light-
humans are scarce. The cyanobacterium Arthrospira (Spi- acclimated state of the organism, nutrient availability, and
rulina) was collected by the ancient Aztecs in Mexico as a metabolite accumulation. Each system needs to be opti-
food additive. In some regions (e.g., Chad in Africa or mized for its specific purpose; there is no universal,
Myanmar in Asia) smaller amounts are still harvested from all-purpose photobioreactor [26, 63]. The choice of a
natural populations in alkaline subtropical lakes. At pres- suitable cultivation system and the adjustment of the cul-
ent, the bulk of microalgal biomass—about 8,000 metric tivation regime must be worked out for each individual
tons used for biotechnological purposes annually—is pro- production strain.
duced extensively in cultivation units, where cultures are Thousands of microalgal strains have been isolated from
exposed to light with sufficient mixing and gas exchange natural habitats and are kept in numerous culture collec-
(autotrophically), or alternatively grown on organic sub- tions around the world. However, only a few strains,
strates as a source of carbon and energy (mixotrophic or mostly of aquatic origin, have been cultivated in large-
heterotrophic cultivation). scale production systems of hundreds to thousands of liters.
Numerous cultivation systems have been designed for A list of strains and their use is shown in Table 1.
growth of microalgae since the 1940s. In general, they are Arthrospira (Spirulina) platensis is a planktonic fila-
optimized to suit a certain strain, purpose or product. mentous cyanobacterium composed of individual cells
Basically, two approaches to mass culturing of microalgae (about 8 lm in diameter) that grows in subtropical alkaline
for the purpose of biomass production exist: the first lakes with a temperature optimum of about 35°C. In pro-
applies to cultivation in large-area open reservoirs (ponds ductive cultures, Arthrospira is cultivated in shallow mixed
and raceways), while the second represents closed vessels, ponds or semiclosed tubular photobioreactors in inorganic
i.e., photobioreactors2 or fermentors; for review, see [61, salts with high concentration of bicarbonate, keeping pH
78]. The first type—open cultivation systems—is repre- above 9. Its biomass is widely used as a health food and
sented by natural or artificial ponds, raceways (ponds akin feed supplement containing proteins, fatty acids, phyco-
to racetracks), and cascades (i.e., inclined-surface systems). biliproteins, carotenoids, polysaccharides, vitamins, and
The second type—photobioreactors (closed or semiclosed minerals.
systems with natural or artificial illumination)—consist of The microalga Chlorella (green algae Chlorophyta) is a
glass or transparent plastic tubes, or panels, positioned cosmopolitan genus with small globular cells (3–8 lm in
horizontally or vertically, arranged as serpentine loops, diameter), including strains with a broad range of tem-
flexible coils, manifold rows, or ‘‘fences,’’ in which the perature tolerance between 15°C and 40°C. Chlorella
microalgal suspension is continuously circulated [51]. grows autotrophically in an inorganic medium, as well as
Until recently, most large-scale commercial microalgal in mixotrophic and heterotrophic conditions (for example,
production systems employed open systems. However, with addition of acetic acid and glucose). At present,
several large-scale closed systems have been built recently autotrophic production of Chlorella is carried out in open
and, for the first time, comparisons of their performance ponds, semiclosed tubular photobioreactors, or inclined
can be made. There are major operational differences cascades, since its fast growth prevents contamination by
other microalgae. Chlorella is the most cultivated eukary-
2
In this article, the term ‘‘photobioreactor’’ is used for closed or otic alga, since it is widely used as a health food and feed
semiclosed systems using natural or artificial illumination. supplement, as well as in the pharmaceutical and cosmetics

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J Ind Microbiol Biotechnol (2010) 37:1307–1317 1309

industries. It contains proteins, carotenoids, some immu- with continuous circulation of the culture by a pump and
nostimulators, polysaccharides, vitamins, and minerals. supply of CO2 to promote growth. The highest biomass
The bulk of the microalgal biomass market is represented concentration achieved was 1.5 g DW l-1. In Israel, a
by Chlorella and Arthrospira, with annual production of small-scale pilot plant to produce biomass of Chlorella or
3,000 and 4,000 t, respectively. Scenedesmus as green fodder for cattle was set up as a
Hypersaline strains of the genus Dunaliella have cells closed, mixed reservoir of 120 l mounted in a greenhouse
about 10 lm in diameter. This microalga produces b-car- and taking advantage of climatic conditions with year-
otene in large amounts, and it is a natural source of round sunlight availability [23]. In Germany, Gummert and
carotenoids for some shrimps. The high content of b-car- coworkers experimented with large-scale cultures of
otene makes Dunaliella attractive to biotechnologists for Chlorella grown in deep shallow concrete trenches (res-
large-scale production in shallow, open ponds under high ervoirs) with plastic lining [27]. These experiments were
solar radiation. aimed at evaluating the possibility of utilizing carbon
Haematococcus pluvialis (Chlorophyta) is a freshwater, dioxide from waste gases in the industrial district of the
unicellular alga with a rather complex lifecycle. A two- Ruhr. The cultures were bubbled with a mixture of air and
stage process is employed for biomass production. Under 1% commercial or ‘‘waste’’ CO2, which was sufficient to
stress conditions (nutrient deficiency, salinity, high tem- supply carbon and maintain growth as well as to mix the
peratures in combination with high irradiance), it produces cultures turbulently.
an orange–red pigment, astaxanthin, the important natural In Japan, at the Tokugawa Institute of Biological
colorant for salmonid fish, shrimp, lobster, and crayfish and Research, an early attempt was made to design a relatively
for the health food market. well-controlled outdoor closed system: a tubular photobi-
oreactor to study the growth kinetics of Chlorella [75]. The
Initial period of microalgal mass culture 40-l system consisted of a horizontal loop of glass tubes
(diameter 3 cm, length 33 m) and was submerged in a
Mass cultivation of microalgae was pioneered by the water bath to prevent overheating. The culture was circu-
Carnegie Institution in the 1940s, and particular attention lated by a pump and aerated by CO2-enriched air, and the
was paid to the unicellular green microalga Chlorella produced oxygen was released in a gas exchange tower.
pyrenoidosa (Chlorophyta) because of the broad range of The Chlorella culture was grown in batch or semibatch
environmental conditions under which it can grow, and regimes with daily harvest of biomass. In the same labo-
also because this organism was then extensively used as a ratory, a ‘‘partially enclosed atmosphere system’’ was
model organism for basic photosynthetic research (see constructed using shallow troughs covered with plastic
pioneering works by Bessel Kok, Melvin Calvin, Robert sheets [55]. The advantage of this system was its relatively
Emerson, and others). The main aim was to obtain high- small depth (2–15 cm) with high linear velocity of
protein biomass and to study its possible uses. During suspension flow (6–45 cm s-1), which guaranteed high
World War II, the experience gained in the cultivation of turbulence of the culture. From the point of view of
Chlorella was applied, namely in the search for an anti- hydraulics, this system did not differ from the open sys-
bacterial substance that might be isolated from the culture tems, since the microalgal culture absorbed sunlight and
of Chlorella [1]. In 1947–1948, the possibility of growing exchanged gases with the atmosphere through its surface.
Chlorella on a large scale for food was seriously consid- In addition, the cultures were supplemented by overhead
ered [10]. The primary cultivation studies of several artificial illumination. Soon after, the technology of Chlo-
research groups were summarized in the ‘‘bible’’ of early rella cultivation for food and feed was worked out in more
algal biotechnology, edited by John S. Burlew of the detail [74].
Carnegie Institution in Washington, DC [9]. The first
attempts at large-scale microalgal cultivation and design of Microalgal biotechnology in Czechoslovakia
early pilot plants focused on closed systems in order to
isolate cultures from the natural environment. This was a By the mid 1950s it was proven that open outdoor cultures
logical consequence of the requirement for controlled were feasible and that they would probably not suffer from
growth conditions and to prevent contamination of cultures contamination more than any large-scale closed culture
by other microorganisms. One of the first productive pilot system if fast-growing strains (e.g., Chlorella or Scene-
plants for mass cultivation of Chlorella was devised and desmus) were cultivated. This is understandable if one
tested at Arthur D. Little, Inc. in Cambridge, MA in col- realizes that it is practically impossible to keep large
laboration with the Carnegie Institution in 1951 [3]. The installations under sterile conditions. Once the feasibility of
cultivation unit (*4,000 l) was constructed from thin- open cultures was confirmed, the concept of outdoor mic-
walled plastic tube to form a flat channel of 7–8 cm depth roalgal culture was substantiated. This is without doubt due

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Fig. 2 Pilot algal outdoor cultivation units constructed in 1958 at the

Botanical Garden in Košice (Slovakia)
Fig. 1 Dr. Ivan Šetlı́k (1928–2009), pioneer and leading figure of
algal biotechnology and photosynthesis research in the former
Czechoslovakia

to the much simpler design and inexpensive construction of

open-type units as compared with closed systems.
With this in mind, the first experimental outdoor units
for cultivation of microalgae in the former Czechoslovakia
were built at the Botanical Garden of the Slovak Academy
of Sciences in the town of Košice at the end of the 1950s.
The research group there was headed by plant physiologist
Fig. 3 Schematic diagram of cascade cultivation unit of 12 m2. The
Ivan Šetlı́k (Fig. 1) with interest in factors limiting plant cultivation surface was set up as shallow troughs made of reinforced
productivity. Initially, the study of microalgae was only a polyester resin and arranged stepwise (1960)
minor research topic, but soon the potentially high pro-
ductivity of photosynthetic microorganisms was realized South Bohemia. Here Ivan Šetlı́k and his collaborators
and became the main focus of laboratory research [5, 6, enjoyed ample support and soon, working with enormous
72]. In 1958, the short popular-science movie Solar Lab- enthusiasm, they developed a number of indoor and out-
oratory was filmed at the Košice Botanical Garden. The door test production units as well as cultivation procedures
film producer, Miro Bernat, liked the idea that microalgal (Fig. 4). Both Ivan Málek and Ivan Šetlı́k (Fig. 5) played
cultivation units would be ‘‘fields for the third millennium’’ decisive roles in the formation and establishment of algal
and financed construction of the first larger microalgal pilot biotechnology in the former Czechoslovakia: throughout
units (Fig. 2). These ‘‘movie’’ units demonstrated well the his whole career, Ivan Šetlı́k (1928–2009) was a visionary
potential for outdoor photosynthetic production of micro- who set research directions not only in algal biotechnology
algal biomass in Central European climate conditions. The but in photosynthesis in general, while Ivan Málek
first cultivation units were based on the principle of the (1909–1994) in the 1960s provided the necessary back-
descending flow surface, constructed as shallow troughs of ing—infrastructure, support, and international promotion.
reinforced polyester resin, arranged stepwise one below The new research group in Třeboň was formed on the
another to form a cascade of hydraulic jumps (Fig. 3, principle of a complex processes approach to study mic-
originally in [73]). A few months later, these units also roalgal productivity; research interests included mathe-
caught the attention of Professor Ivan Málek, then director matical modeling of turbulent flow, instrumentation
of the Institute of Biology in Prague, who visited Košice. development, biotechnology, physiology, cell biology,
He recognized the potential role of mass microalgal culti- algal genetics, and ecophysiology. Soon after, the micro-
vation in the broader aim of managing continuous culti- algal biotechnology research was moved to a new labora-
vation of industrial microorganisms and providing tory campus ‘‘Opatovický mlýn,’’ reconstructed from the
alternative sources of protein. With his assistance, the former watermill built in 1708 by Augustinian priests.
Algological Laboratory of the Institute of Biology was There, research was aimed at defining the scientific basis
established in January 1960 in Třeboň, a small town in for commercial exploitation of microalgae cultivated on a

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J Ind Microbiol Biotechnol (2010) 37:1307–1317 1311

Fig. 6 Semiproduction outdoor algal production units located in the

campus of the Opatovický mlýn (mid 1960s)
Fig. 4 Test outdoor cultivation units operating in Třeboň (early
1960s) in a relatively thin layer (1–5 cm). This guarantees high
average irradiance per cell and good gas exchange to obtain
higher productivity per illuminated surface. Thus, a much
lower volume of dense microalgal suspension can be
treated at harvest. The units had a plane glass surface, with
a slope of 3%, supported by a steel structure. The surface
was fitted with transverse baffles 3.5 cm high and 15 cm
apart to create intensive turbulence in the microalgal sus-
pension, which moved down the surface at a velocity of
7 cm s-1. The culture was circulated over the surface by an
axial-flow pump during the day and was kept in a retention
tank at night to reduce heat losses, or during rainfall to
avoid dilution by rainwater. The detailed setup of these
unique cultivation experiments was described in a newly
established journal, Algological Studies, which was initi-
ated by Prof. Málek and published in Třeboň [47, 71]. In
outdoor cultivation experiments as well as in most labo-
Fig. 5 Ivan Šetlı́k (left) and Ivan Málek (right) celebrating the first ratory studies, the green microalga Scenedesmus quadric-
successful cultivation of microalgae in Třeboň (early 1960s) auda with large coenobia which settle easily, and later
Chlorella pyrenoidosa with small, globular cells (2–8 lm
large scale. In 1962–1963, unique outdoor pilot plants of in diameter), were used. In the 1960s, the efforts of the
50 and 900 m2 were constructed [73]. The original highly laboratory were focused mostly on the technical problems
productive units were based on cascades of sloping planes, of microalgae mass culture.
known worldwide as Třeboň-type cascade units (Fig. 6). Later, in the 1960s and 1970s, cascade thin-layer culti-
The principle of microalgal cultivation was to maintain vation units of Třeboň type were also constructed in
turbulent flow of a relatively thin layer using corrugated Poland, Cuba, Bulgaria, and Italy to compare cultivation
surfaces or a plain surface fitted with baffles. The pilot under various climatic conditions [71, 79, 87]. Collabora-
plant was constructed as dual-purpose units that were used tion was also established with the biotechnology group of
during the winter as a glasshouse for hydroponic vegetable the Istituto di Microbiologia Agraria, Universita di Firenze,
and flower cultivation and in summer for microalgal bio- which worked with mass microalgae cultures [24, 25].
mass production. A project for a large algal production plant and research
Compared with reservoirs (open ponds, raceways), with institute was elaborated in the late 1960s. The plant was to
suspension depth of 20–30 cm where dilute cultures of be situated on the opposite side of the Opatovický pond. The
microalgae (0.5-1 g DW l-1) were grown under limited proposed layout combined a research and development
mixing and gas exchange, the main advantage of the cas- (R&D) center with biomass production, to create optimal
cade system constructed in Třeboň was the growth of a conditions for fast and flexible solution of all problems
well-mixed thick (10–15 g DW l-1) microalgal suspension which are to be expected during the scale-up process [71].

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However, the plant and the institute never materialized, and circulated by paddlewheels with a culture layer twice as deep
even the existing pilot units were dismantled after only a few (17.2 g DW m-2 day-1) when working with the green
years of operation. Following the initial enthusiasm, a cer- microalga Scenedesmus obliquus [4]. Due to the different
tain stagnation in algal research followed, and skepticism culture concentration, areal densities in terms of algal
about the economical potential of algal mass production biomass per unit surface were equivalent, but the greater
took over. In fact, several pilot plants that started operation turbulence and better temperature regime of the cascades led
in the 1960s and 1970s failed to confirm the hopeful pros- to their higher productivity as compared with the raceways.
pects derived from the earlier laboratory work of the pre- At the beginning of the 1990s, a third generation of out-
vious decades. Most of the large-scale tests were abandoned door cascade units for microalgal cultivation was built in
after a few months of operation with the conclusion that, for Třeboň. Compared with the cascades used in the 1960s, the
the time being, large-scale culture could not be economi- microalgal suspension in the new cascade units was much
cally feasible. The curious thing about this situation is that in thinner—only about 10 mm thick. Instead of densely spaced
no case was a reasonable explanation given for the diver- baffles as described previously [71], plastic rods of 13 mm
gence between the conclusions drawn from basic research diameter were placed 1.5 meters apart, and thus the flow
and the results obtained with pilot-plant equipment. The velocity could be increased to 0.5 m s-1 [17, 18]. Later, it
only explanation which seemed reasonable was imperfec- was realized that the inclined-surface system works best if
tions of the technical equipment and technology; in fact, as operated as a smooth inclined surface without any baffles
was often stated, the conditions obtained for growth of where the layer of microalgae is only about 6 mm [44]. This
microalgae were rather distant from the optimal ones [73]. allows achievement of high growth rate up to biomass
However, the main reason for dismantling the pilot concentration of 40–50 g DW l-1. Also, cleaning and main-
production plant in Třeboň was that, after the political tenance were much simpler compared with the baffled
turmoil in 1968, Professor Málek was removed from office system. A 50 m2 pilot system was tested in the Mediterranean
as a ‘‘revisionist,’’ and every activity, even scientific, that climate where summer productivities were as high as
he had supported was intentionally suppressed. Partial 32 g DW m-2 day-1, as compared with Central Europe with
resurrection of microalgal biotechnology in Třeboň came productivity maximum of about 23 g DW m-2 day-1 [16].
only at the end of the 1970s, owing to the role of Ivan Important issues relating to construction design, variation
Šetlı́k and his collaborators in the space program ‘‘Inter- of cultivation regimes, and biomass productivity studies
cosmos,’’ in which they prepared experiments for a Czech were addressed by measurements of pCO2 and pO2 profiles
astronaut on board the Salyut 6 spacecraft that orbited in in outdoor cascade units carried out by Karel Lı́vanský and
1978. The novel experiments were successful and proved co-workers [38, 45]. In some experiments, natural gas from
that unicellular Chlorella can grow and divide under an underground source was used for cultivation [44, 45].
microgravity conditions of space shuttles [36]. These measurements provided detailed results on CO2/O2
exchange to optimize the supply of CO2 for microalgal mass
cultures and its utilization in thin-layer open units with long
Microalgal photosynthesis and biotechnology after 1989 cultivation tracks [37, 39–41, 43]. About 64% of supplied
CO2 was utilized by the microalgal culture, the rest being
After the return of democracy in 1989 any direct political lost as a result of incomplete absorption in the process of
influence on Czech science was removed. In the two dec- saturation or escape from the suspension into the atmo-
ades which followed, enormous development in the field of sphere. About 2.73 kg CO2 was needed for production of
algal biotechnology occurred. In September 1993, the 6th 1 kg Chlorella biomass. Per 1 g evolved O2, 1.12 g CO2
International Conference on Applied Algology was held at was consumed by the microalgae [42].
Třeboň [2]. It opened new topics and renewed collabora- Based on fundamental research into the structure and
tions broken at the end of the 1960s. At this time, a great function of photosynthetic membranes carried out at the
renaissance of microalgal biotechnology also occurred Laboratory of Photosynthesis in Třeboň in the 1980s and
worldwide. New applications were discussed and new 1990s [56, 57], microalgal biotechnology moved from a semi-
approaches were designed. empirical to molecular level. New methodology of photo-
biophysical and biochemical measurements was employed.
Before the turn of the millennium Laboratory cultures of cyanobacteria and microalgae were
examined to define the role of photosystem II (PSII) complex
In the 1980s, it was proven experimentally that cascades in response to environmental stresses [30, 32, 33].
(inclined baffled surface with about 3 cm culture layer) cir- Since the early attempts in the 1930s, it has been clear
culated by a pump could achieve significantly higher pro- that intermittent (pulsed) light is the most important factor
ductivity (24.8 g DW m-2 day-1) than horizontal raceways for microalgae growth. The amount of photon energy

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received by each cell is a combination of several factors: or higher depression of photochemical yields indicates low-
irradiance intensity, cell population density, length of light-acclimated or photoinhibited cultures, respectively.
optical path (thickness of culture layer), spectral quality, These results are important from a biotechnological point of
light absorption, and rate of mixing [63]. Turbulent regime view in order to optimize the growth of outdoor microalgal
in mass microalgal cultures is essential, since light/dark mass cultures under varying climatic conditions.
cycles determine culture productivity. Short pulses of high The so-called xanthophyll cycle (light-dependent con-
light intensity can be used with high efficiency if separated version of violaxanthin to zeaxanthin), was shown to serve as
by sufficiently long dark periods [22]. Maximal quantum a major, short-term light-acclimation mechanism in higher
yields were found for light/dark ratios of about 1:10 [31]. plants. The role of xanthophylls in thermal dissipation of
In pulsed (intermittent) light regimes, a microalgal culture surplus excitation energy was deduced from the linear
can utilize a larger fraction of the sunlight reaching a given relationship between zeaxanthin formation and the magni-
area. In the 1990s, the introduction of high-intensity light- tude of nonphotochemical fluorescence quenching. Unlike
emitting diodes (LEDs) for scientific use made it possible in higher plants, the role of the xanthophyll cycle in green
to measure the effect of intermittent illumination more microalgae (Chlorophyta) is ambiguous, since its contribu-
preciously in the microsecond range. It was proved that tion to energy dissipation can vary significantly among
photosynthetic rates could be further enhanced if the fre- species [48, 52]. It was found that the xanthophyll cycle
quency of intense light pulses was increased from units to operates in all tested strains (e.g., Chlorella, Scenedesmus,
thousands of Hz [26, 54, 58]. Lately, a hydrodynamic Haematococcus, Chlorococcum, Spongiochloris); however,
model of culture in thin-layer cascade units has demon- its contribution to nonphotochemical quenching was not as
strated highly turbulent flow allowing rapid light/dark significant as in higher plants. It seems that microalgae rely
cycles (with frequency of 0.5 s-1) [51]. on this dissipative mechanism only at low biomass density.
Following the application of chlorophyll (chl) variable Another new line of research was focused on microalgal
fluorescence measurements in field crops with the aim of secondary metabolites. The search was for potential pro-
correlating changes of photobiochemical activities with ducers of secondary carotenoids, extracellular polysaccha-
productivity (e.g., [13]), chl fluorescence was applied to rides, mycosporine-like amino acids, and polyunsaturated
monitor microalgal mass cultures in situ. This novel fatty acids. Beside isolation and characterization of these
approach was qualitatively different from previously used compounds, research also aimed to optimize parameters
physiological methods, and the photosynthetic and bio- important for the design and construction of suitable labo-
technology groups from Třeboň played a substantial role in ratory photobioreactors that would be suitable for overpro-
its development. The pilot experiments, carried out in cas- duction of these bioactive compounds on the scale of
cades and closed photobioreactor systems in the Czech hundreds of liters [29, 34, 35, 65, 86].
Republic, Italy, and Israel, mostly applied the method of chl Because phototrophic microalgae can be cultivated
fluorescence quenching to examine effects of environmental under strictly controlled conditions, they are the ideal
stresses—high irradiance, temperature extremes, high dis- choice to incorporate stable isotopes from inorganic C, H,
solved oxygen concentration, and their synergism on algal and N sources. Various biochemicals labeled by stable
productivity [76, 81]. Online chl fluorescence measure- isotopes are used for scientific purposes (molecular struc-
ments indicated that changes of daily integrated values of ture or physiological investigations), as well as for clinical
relative PSII electron transport could be correlated well with purposes (gastrointestinal or breath diagnosis tests) [15].
analogous changes in daily productivity of cultures grown
under different conditions [52, 53, 77]. The relative electron Microalgal biotechnology in the third millennium:
transport rate proved to be a simple and reliable parameter future prospects
for use in estimating the photosynthetic performance of
outdoor cultures of microalgae. Thus, in situ chl fluores- Since 2000, close collaboration with the Academic and
cence monitoring has proven to be a suitable technique for University Center in Nové Hrady resulted in the construction
measuring photochemical performance, being fast, nonin- of a brand new type of closed tubular photobioreactor which
vasive, and easy to measure. However, although the theory was based on solar concentrators (linear Fresnel lenses)
is well described at present [66, 70, 82, 83], the interpreta- mounted in a climate-controlled greenhouse on top of the
tion of fluorescence signals may not be straightforward, laboratory complex, combining features of indoor and out-
particularly when dealing with microalgae [11, 67, 69]. door cultivation units [49, 50]. The dual-purpose system was
Experiments in closed photobioreactors as well as open designed for algal biomass production in temperate climate
cascade units showed that a midday depression of PSII zone under well-controlled cultivation conditions and with
photochemical yields of 20–30% of maximal morning values surplus solar energy being used for heating service water. It
is essential for well-performing cultures [49–51, 63]. Lower was used to study the strategy of microalgal acclimation to

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supra-high solar irradiance, with values as much as 3.5 times Particularly, the search for novel anti-inflammatory
the ambient value. In model cultivations, cultures of the substances, able to downregulate increased endothelial
cyanobacterium Arthrospira (Spirulina) were cultivated at chemokine production and adhesion molecule expression as
about three times higher solar irradiances (as high as 6 mmol well as tissue damage, holds therapeutic promise [46]. In
photon m-2 s-1) than those usually recorded outdoors in close collaboration with Austrian partners (IMC Krems),
summer, indicating that this organism is tolerant to pho- two cell lines were used for detection of anti-inflammatory
toinhibition under sufficient turbulence and biomass density. and wound-healing metabolites from microalgae [85]. The
A two-stage cultivation process of the green microalga use of new in vitro assays resulted in detection of several
Haematococcus pluvialis was investigated with respect to compounds with unique structures and potential as novel
correlations between photochemical activities and asta- therapeutics. The search for new acetylcholine esterase
xanthin production. First, the culture was grown in low- inhibitors was successful using primary screening of more
irradiance units, and then exposed to supra-high irradiance than 200 microalgal strains from different habitats during
when the rate of astaxanthin production was 30–50% this period of time [89]. Compounds isolated from the
higher than in the culture exposed to ambient irradiance. cyanobacterium Nostoc spp. were structurally characterized
Carotenoid-rich microalgae biomass can be used as a col- [14, 90]. In the field of antioxidant activity a new rapid-
orant, for example, in ornamental fish aquaculture [88]. resolution separation method was developed [59]. The
The light captured by photosynthetic pigments is roughly method was optimized for determination and identification
ten times higher under full sunlight (2,000 lmol photon of antioxidants (phenolic compounds and isoflavones) in
m-2 s-1) than that required to saturate growth. In other fmol quantities and submicroliter sample volumes; for
words, as much as 90% of the photons captured by chl example, p-hydroxybenzoic, protocatechic, vanillic, syrin-
antennae are dissipated as heat and fluorescence. Uncritical gic, caffeic, and chlorogenic acids, 4-hydroxybenzaldehyde,
acceptance of photosynthetic efficiencies of about 10% or and 3,4-dihydroxybenzaldehyde were identified in extracts
even higher [60] inevitably leads to exaggerated estimates from microalgae strains (i.e., Spirulina platensis, Anabaena
of present and future biomass productivity. We can estimate doliolum, Nostoc spp., and Cylindrospermum spp.) which
a more realistic figure for maximum photosynthetic effi- contain phenolic acids or aldehydes at ppt levels.
ciency (photon energy converted into biomass energy) of Another promising field in current microalgae research
about 4.5% for C3 plants or microalgae by using educated is the quest for biofuels. As the rate of fossil-fuel con-
guesswork and detailed consideration of the partial reac- sumption increases to unsustainable levels and accumula-
tions involved (e.g., [7, 8, 84, 91]). tion of greenhouse gases in the environment quickly
A new type of microalgal bioreactor with precise control approaches ‘‘dangerously high’’ concentrations, a new
of process parameters (temperature, irradiance, gas com- bonanza for microalgal biotechnology has started with the
position) and online measurement of photosynthetic per- goal of economical biofuel production from microalgae. A
formance based on chl fluorescence was designed [12] and brief overview of second-generation biodiesel production
are now produced commercially (www.psi.cz). In parallel systems using microalgae has been compiled [64]. To
with the design of the new generation of photobioreactors, achieve environmental and economic sustainability, fuel
the study of bioactive compounds in microalgal cultures production processes are required that are not only
with potential as pharmacological drug leads has been renewable but also capable of sequestering atmospheric
initiated. The natural product chemistry is extraordinarily CO2. Biodiesel is currently produced from oil synthesized
diverse, reflecting the exceptional biosynthetic capacities from conventional fuel crops that harvest the Sun’s energy
of microalgae. The molecular targets relevant for drug and store it as chemical energy. This presents a route for
discovery have been generated by nature for millennia, but renewable and carbon-neutral fuel production. However,
the technical knowhow to isolate and characterize bioactive increasing biofuel production on arable land could have
compounds has only been available recently [68]. Micro- severe consequences for global food supply. Second-gen-
algae represent a large, unexplored source of a variety of eration biofuels (biodiesel, bioethanol, and biomethane)
chemical structures. Routine methods for evaluation of produced from microalgae, plant, and forest plantations on
biological activity in extracts from microalgal biomass as vast land areas will have the advantage that they do not
well as culture media have been applied. Lately, new test compete with food crops. However, current supplies from
systems to identify drug candidates were applied for cell- oil crops and animal fats account for only approximately
based primary screening of several hundred microalgae 0.3% of current demand for transport fuels. In contrast to
strains for antibacterial, antioxidant, fungicidal, allelo- fuel crops, producing biodiesel from algae is widely
pathic, antitumoral, wound-healing, and anti-inflammatory regarded as one of the most efficient ways of generating
compounds as well as enzyme (proteases, acetylcholine biofuels and also appears to represent the only current
esterase) activity inhibitors [21, 28, 46, 59, 80, 85, 89, 90]. renewable source of oil that could meet global demand for

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transport fuels. The main advantages of second-generation 2. Algal Biotechnology (1993) Progress in Biotechnology of
microalgal systems are that they: (1) have a high photon Phototrophic Microorganisms. 6th international conference on
applied algology, České Budějovice, p 155
conversion efficiency (as evidenced by increased biomass 3. Arthur DL (1953) Pilot plant studies in the production of Chlo-
yields per hectare), (2) can be harvested batch-wise nearly rella. In: Burlew JS (ed) Algal culture: from laboratory to pilot
all year round, providing a reliable and continuous supply plant. Kirby Lithographic, Washington, DC, pp 235–272
of oil, (3) can utilize salt and waste water, thereby greatly 4. Balloni W, Materassi R, Pelosi E, Pushparaj B, Florenzano G,
Stengel E, Soeder CJ (1981) Comparison of two different culture
reducing freshwater use, (4) can couple CO2-neutral fuel devices for mass production of microalgae at Firenze (Italy) and
production with CO2 sequestration, and (5) can produce Dortmund (Germany). Algol Stud Arch Hydrobiol 28:324–331
nontoxic and highly biodegradable biofuels. Current limi- 5. Bartoš J (1959) Pestovanie rias v laboratórnych a produkčných
tations exist mainly in the harvesting process and in the podmienkach. Naša veda 6:155–158
6. Bartoš J, Šetlı́k I (1959) Laboratorné a prevádzkové zariadenia na
supply of CO2 for high-efficiency production. Preliminary pestovanie rias. Naša veda 6:360–364
studies were carried out on utilization of CO2 from flue 7. Benemann JR, Oswald WJ (1996) Systems and economic anal-
gasses for cultivation of microalgae in outdoor open thin- ysis of microalgae ponds for conversion of CO2 to biomass, Final
layer units [19] or in closed cultivation systems [20]. A report. US DOE
8. Boardman NK (1980) Energy from the biological conversion of
scheme for a combined process of farm unit size was solar energy. Philos Trans R Soc Lond A Math Phys Eng Sci
proposed; this includes anaerobic digestion of organic 295:477–487
agricultural waste, production and combustion of biogas, 9. Burlew JS (1953) Algal culture: from laboratory to pilot plant.
and utilization of flue gas for production of microalgal Kirby Lithographic, Washington, DC
10. Burlew JS (1953) Current status of the large scale culture of
biomass, which could be used in animal feed. algae. In: Burlew JS (ed) Algal culture: from laboratory to pilot
Throughout the last 50 years, microalgal biotechnology plant. Kirby Lithographic, Washington, DC, pp 3–23
has undergone enormous development. In the former 11. Campbell D, Hurry V, Clarke AK, Gustafsson P, Oquist G (1998)
Czechoslovakia, the foundations of microalgal biotech- Chlorophyll fluorescence analysis of cyanobacterial photosyn-
thesis and acclimation. Microbiol Mol Biol Rev 62:667–683
nology were laid in the 1960s by a group of enthusiasts that 12. Červený J, Šetlı́k I, Trtı́lek M, Nedbal L (2009) Photobioreactor
included Prof. Ivan Málek. Despite the fact that research for cultivation and real-time, in-situ measurement of O2 and CO2
and practice of microalgal biotechnology had to pass exchange rates, growth dynamics, and of chlorophyll fluores-
through difficult and critical periods, the foundations cence emission of photoautotrophic microorganisms. Eng Life
Sci 9:247–253
proved to be solid. Today, several original questions 13. Corlett JE, Jones HG, Masojidek JM, Massacci A (1992) Chlo-
remain unanswered and many visions still need to be ful- rophyll fluorescence in the field-grown sorghum—instrument
filled. Despite the fact that research and biotechnological discrepancies. Photosynthetica 27:257–260
priorities have changed, in that microalgae are not con- 14. Čejka J, Kopecký J, Lukešová A, Štys J, Zelı́k P (2008) The
cyanobacteria strain Nostoc Lukešová 27/97 and the procedure
sidered primarily as the source of proteins or vitamins, but for isolation of acetylcholine esterase inhibitor. Patent No.
rather of carbon storage products to generate clean energy, 299567, Czech Republic
the major underlying questions of microalgal biotechnol- 15. Doucha J, Kopecký J, Tintěra Š, Smažı́k M (1990) Cultivation of
ogy are still ‘‘hot’’ and open for further research: What is algae and cyanobacteria for biosynthesis of 13C-labelled com-
pounds and the unit for this procedure. Patent No. 276540, Czech
the maximal possible microalgal productivity, and how can Republic
it be achieved on a large scale and in an economic way? 16. Doucha J, Livansky K (2006) Productivity, CO2/O2 exchange and
Can microalgal cultures accumulate high content of carbon hydraulics in outdoor open high density microalgal (Chlorella
storage products and still grow rapidly? Let us hope that sp.) photobioreactors operated in a Middle and Southern Euro-
pean climate. J Appl Phycol 18:811–826
modern approaches will provide positive answers. 17. Doucha J, Lı́vanský K (1995) Novel outdoor thin-layer high
density microalgal culture system: productivity and operational
Acknowledgments The authors thank Dr. Jiřı́ Kopecký for valuable parameter. Algol Stud Arch Hydrobiol 76:129–147
discussion of the manuscript. The Czech Academy of Sciences sup- 18. Doucha J, Lı́vanský K, Bı́nová J, Kubičko P, Novotný P (1993)
ported this work through the Institutional Research Concept Thin-layer high density microalgal culture system: productivity
AV0Z50200510 ‘‘Microorganisms in Research and Biotechnology.’’ and operational energy costs. Progress in Biotechnology of
Partial funding was also provided by the Czech Science Foundation Phototrophic Microorganisms. Proc. 6th internatinal conference
project GACR 521/09/0656. on applied algology, Třeboň, Czech Republic, p 40
19. Doucha J, Straka F, Livansky K (2005) Utilization of flue gas for
cultivation of microalgae (Chlorella sp.) in an outdoor open thin-
layer photobioreactor. J Appl Phycol 17:403–412
20. Doušková I, Doucha J, Lı́vanský K, Machat J, Novák P, Umysová
References D, Zachleder V, Vı́tová M (2009) Simultaneous flue gas biore-
mediation and reduction of microalgal biomass production costs.
1. Pratt R, Daniels TC, Eiler JJ, Gunnison JB, Kumler WD, Oneto Appl Microbiol Biotechnol 82:179–185
JF, Strait LA, Spoehr HA, Hardin GJ, Millner HW, Smith JHC, 21. Drápalová P, Štys J, Lukešová A, Kopecký J (2008) Genus
Strain HH (1944) Chlorellin, an antibacterial substance from Nostoc—a source of novel trypsin inhibitors. Algol Stud Arch
Chlorella. Science 99:351–352 Hydrobiol 127:232–241

123
1316 J Ind Microbiol Biotechnol (2010) 37:1307–1317

22. Emerson R, Arnold W (1932) A separation of the reactions in 41. Lı́vanský K, Doucha J (2003) Evaluation of dissolved oxygen
photosynthesis by means of intermittent light. J Gen Phys (DO) profiles in microalgal suspension on outdoor thin-layer
15:391–420 cultivation surface. Algol Stud Arch Hydrobiol 110:151–165
23. Evenari M, Mayer AM, Gottesman E (1953) Experiments on 42. Lı́vanský K, Doucha J (2005) Utilization of carbon dioxide by
culture of algae in Israel. In: Burlew JS (ed) Algal culture: from Chlorella kessleri in outdoor open thin-layer culture units. Algol
laboratory to pilot plant. Kirby Lithographic, Washington, DC, Stud Arch Hydrobiol 116:201–212
pp 197–203 43. Lı́vanský K, Doucha J, Hu HJ, Li YG (2006) CO2 partial pres-
24. Florenzano G (1963) La coltura massiva di alghe. Enciclopedia sure—pH relationships in the medium and relevance to CO2 mass
delle Science e delle Tecnice ‘‘Il Leonardo’’ 51:465–472 balance in outdoor open thin-layer Arthrospira (Spirulina) cul-
25. Florenzano G, Balloni W, Materassi R (1960) Un triennio di tures. Arch Hydrobiol 165:365–381
sperimentazione italiana sulla coltura massiva delle alghe 44. Lı́vanský K, Kajan M, Pilarski PS (1995) Productivity, respira-
microscopiche nell’impianto pilota di Firenze. Institute Micro- tion and chemical composition of the green alga Scenedesmus
biologia Agraria e Technica-Universita di Firenze, Firenze, p 46 incrassatulus grown in outdoor cultivation units with and without
26. Grobbelaar JU (2009) Factors governing algal growth in photo- baffles. Algol Stud Arch Hydrobiol 76:111–128
bioreactors: the ‘‘open’’ versus ‘‘closed’’ debate. J Appl Phycol 45. Livansky K, Pilarski PS (1993) Carbon dioxide supply to algal
21:489–492 cultures. 2. Efficiency of CO2 absorption from a natural gas
27. Gummert F, Meffert ME, Stratman H (1953) Nonsterile large supplied to the recirculation pipe of a cultivation unit. Algol Stud
scale culture of Chlorella in greenhouse and open air. In: Burlew Arch Hydrobiol 69:113–123
JS (ed) Algal culture: from laboratory to pilot plant. Kirby 46. Madlener S, Svačinová J, Kitner M, Kopecký J, Eytner R,
Lithographic, Washington, DC, pp 235–272 Lackner A, Than Phuong Nha V, Frisch R, Grusch M, de Martin
28. Hrouzek P, Kopecký J, Salát J, Maršálek B, Lukešová A (2005) R, Doležal K, Strnad M, Krupitza G (2009) In vitro anti-
Cytotoxic effect of soil cyanobacterial extracts to mammal cell inflammatory and anticancer activities of extracts of Acalypha
lines YAC-1 and WEHI. Czech Phycol 5:79–90 alopecuroidea (Euphorbiaceae). Int J Oncol 35:881–891
29. Klyachko-Gurvich GL, Tsoglin LN, Doucha J, Kopetskii J, 47. Málek I (1970) Preface. Algol Stud Arch Hydrobiol 1:5–6
Shebalina (Ryabykh) IB, Semenenko VE (1999) Desaturation of 48. Masojı́dek J, Kopecký J, Koblı́žek M, Torzillo G (2004) The
fatty acids as an adaptive response to shifts in light intensity. xanthophyll cycle in green algae (Chlorophyta): its role in the
Physiol Plant 107:240–249 photosynthetic apparatus. Plant Biol 6:342–349
30. Knoppová J, Masojı́dek J, Pokorný J (1993) Chlorophyll fluo- 49. Masojı́dek J, Papáček Š, Sergejevová M, Jirka V, Červený J,
rescence quenching caused by inorganic carbon depletion in the Kunc J, Korečko J, Verbovikova O, Kopecký J, Štys D, Torzillo
green alga Scenedesmus quadricauda. Photosynthetica 28: G (2003) A closed solar photobioreactor for cultivation of mic-
541–547 roalgae under supra-high irradiance: basic design and perfor-
31. Kok B (1953) Experiments on photosynthesis by Chlorella in mance. J Appl Phycol 15:239–248
flashing light. In: Burlew JS (ed) Algal culture: from laboratory to 50. Masojı́dek J, Sergejevová M, Rottnerová K, Jirka V, Korečko J,
pilot plant. Kirby Lithographic, Washington, DC, pp 63–75 Kopecký J, Zaťková I, Torzillo G, Štys D (2009) A two-stage
32. Komenda J, Masojı́dek J, Boček J, Prášil O (1993) Reversible and solar photobioreactor for cultivation of microalgae based on solar
irreversible changes of fluorescence parameters during photoin- concentrators. J Appl Phycol 21:55–63
hibition in the Synechococcus elongatus cells. Photosynthetica 51. Masojı́dek J, Torzillo G (2008) Mass cultivation of freshwater
28:249–251 microalgae. In: Jorgensen SE, Fath BD (eds) Ecological engi-
33. Komenda J, Masojı́dek J, Prášil O, Boček J (1992) Two mecha- neering. Encyclopedia of ecology. Elsevier, Oxford, pp 2226–2235
nisms of photosystem 2 photoinactivation—do they exist in vivo? 52. Masojı́dek J, Torzillo G, Kopecký J, Koblı́žek M, Nidiaci L,
Photosynthetica 27:99–108 Komenda J, Lukavská A, Sacchi A (2000) Changes in chloro-
34. Kopecký J (1997) Kinetic model of extracellular polysaccharide phyll fluorescence quenching and pigment composition in the
production by the unicellular red alga Porphyridium purpureum. green alga Chlorococcum sp grown under nitrogen deficiency and
Algol Stud Arch Hydrobiol 87:137–144 salinity stress. J Appl Phycol 12:417–426
35. Kopecký J, Doucha J (1996) Photoadaptation to spectral quality 53. Masojı́dek J, Vonshak A, Torzillo G (2010) Chlorophyll fluo-
of light in the red alga Porphyridium cruentum. Algol Stud Arch rescence applications in microalgal mass culture. In: Suggett DJ,
Hydrobiol 81:53–67 Prášil O, Borowitzka MA (eds) Chlorophyll a fluorescence in
36. Kordyum VA, Shepelev EY, Meleshko GI, Šetlı́k I, Kordyum EL, aquatic sciences: methods and applications. Springer, Dordrecht
Sytnik KM, Mashinsky AL, Popova AF, Dubinin NP, Vaulina 54. Matthijs HCP, Balke H, VanHes UM et al (1996) Application of
EN, Polivoda LV (1980) Biological studies of Chlorella pyre- light-emitting diodes in bioreactors: flashing light effects and
noidosa (strain Larg-1) cultures grown under space flight condi- energy economy in algal culture (Chlorella pyrenoidosa). Bio-
tions. In: Holmquist R (ed) Life sciences and space research. technol Bioeng 50:98–107
Pergamon, Oxford, p 220 55. Mituya A, Nyunoya T, Tamiya H (1953) Pre-pilot-plant experi-
37. Lı́vanský K (2000) Comparison of continuous and stepwise ments on algal mass culture. In: Burlew JS (ed) Algal culture:
control of CO2 supply into outdoor open thin-layer algal culture from laboratory to pilot plant. Kirby Lithographic, Washington,
units. Algol Stud Arch Hydrobiol 96:119–129 DC, pp 273–281
38. Lı́vanský K (1995) Influence of mixing on algal photosynthesis in 56. Nedbal L, Masojı́dek J, Komenda J, Prášil O, Šetlı́k I (1990)
turbulent flow: Modelling approach. Algol Stud Arch Hydrobiol Three types of photosystem II photoinactivation. 2. Slow pro-
78:97–109 cesses. Photosynth Res 24:89–97
39. Lı́vanský K, Doucha J (1997) Additional CO2 saturation of thin- 57. Nedbal L, Šetlı́ková E, Masojı́dek J, Šetlı́k I (1986) The nature of
layer outdoor microalgal cultures: CO2 mass transfer and photoinhibition in isolated thylakoids. Biochim Biophys Acta
absorption efficiency. Algol Stud Arch Hydrobiol 87:145–154 848:108–119
40. Lı́vanský K, Doucha J (1996) CO2 and O2 gas exchange in out- 58. Nedbal L, Tichý V, Xiong FH, Grobbelaar JU (1996) Micro-
door thin-layer high density microalgal cultures. J Appl Phycol scopic green algae and cyanobacteria in high-frequency inter-
8:353–358 mittent light. J Appl Phycol 8:325–333

123
J Ind Microbiol Biotechnol (2010) 37:1307–1317 1317

59. Onofrejová L, Vašı́čková J, Klejdus B, Stratil P, Mišurcová L, 76. Torzillo G, Accolla P, Pinzani E, Masojidek J (1996) In situ
Kráčmar S, Kopecký J, Vacek J (2010) Bioactive phenols in monitoring of chlorophyll fluorescence to assess the synergistic
algae: the application of pressurized-liquid and solid-phase effect of low temperature and high irradiance stresses in Spirulina
extraction techniques. J Pharm Biomed Anal 51:464–470 cultures grown outdoors in photobioreactors. J Appl Phycol
60. Pirt SJ (1986) Tansley review no. 4—the thermodynamic effi- 8:283–291
ciency (quantum demand) and dynamics of photosynthetic 77. Torzillo G, Bernardini P, Masojı́dek J (1998) On-line monitoring
growth. New Phytol 102:3–37 of chlorophyll fluorescence to assess the extent of photoinhibition
61. Pulz O (2001) Photobioreactors: production systems for photo- of photosynthesis induced by high oxygen concentration and low
trophic microorganisms. Appl Microbiol Biotechnol 57:287–293 temperature and its effect on the productivity of outdoor cultures
62. Pulz O, Gross W (2004) Valuable products from biotechnology of of Spirulina platensis (Cyanobacteria). J Phycol 34:504–510
microalgae. Appl Microbiol Biotechnol 65:635–648 78. Tredici M (2004) Mass production of microalgae: photobiore-
63. Richmond A (2004) Biological principles of mass cultivation. In: actors. In: Richmond A (ed) Handbook of microalgal mass cul-
Richmond A (ed) Handbook of microalgal mass cultures. ture. Blackwell Science, Oxford, pp 178–214
Blackwell Science, Oxford, pp 125–177 79. Vendlová J (1969) Outdoor cultivation in Bulgaria. Mass culture
64. Schenk PM, Thomas-Hall SR, Stephens E et al (2008) Second of Scenedesmus in outdoor units. In: Nečas J, Lhotský O (eds)
generation biofuels: high-efficiency microalgae for biodiesel Ann. Rep. Algolog Lab. Třeboň for 1968, pp 143–152
production. Bioenergy Res 1:20–43 80. Voloshko LN, Kopecký J, Safronová T, Pljusch A, Titova N,
65. Schoefs B, Nebesářová J, Kopecký J and Štys D (1998) Pigment Hrouzek P, Drabková V (2008) A variety of toxins produced by
content and location during seed formation in Gleditsia trican- cyanobacteria in Lake Ladoga. Estonian J Ecol 57:1–11
thos. In: Kevers C (ed) Phytohormones, croissance et développ- 81. Vonshak A, Torzillo G, Accolla P, Tomaselli L (1996) Light and
ement, bulletin de la société royale des sciences de liege, 67, 225 oxygen stress in Spirulina platensis (Cyanobacteria) grown out-
66. Schreiber U (2004) Pulse-amplitude-modulation (PAM) fluo- doors in tubular reactors. Physiol Plant 97:175–179
rometry and saturation pulse method: an overview. In: Papa- 82. Vredenberg W, Durchan M, Prášil O (2009) Photochemical and
georgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a photoelectrochemical quenching of chlorophyll fluorescence in
signature of photosynthesis. Advances in photosynthesis and photosystem II. Biochim Biophys Acta 1787:1468–1478
respiration, vol 19. Springer, The Netherlands, pp 279–319 83. Vredenberg W, Kasalický V, Durchan M, Prášil O (2006) The
67. Schreiber U, Endo T, Mi HL, Asada K (1995) Quenching analysis chlorophyll a fluorescence induction pattern in chloroplasts upon
of chlorophyll fluorescence by the saturation pulse method— repetitive single turnover excitations: accumulation and function
particular aspects relating to the study of eukaryotic algae and Of Q(B)-nonreducing centers. Biochim Biophys Acta 1757:
cyanobacteria. Plant Cell Physiol 36:873–882 173–181
68. Skulberg OL (2004) Bioactive chemicals in microalgae. Hand- 84. Walker DA (2009) Biofuels, facts, fantasy, and feasibility. J Appl
book of microalgal mass cultures. In: Richmond A (ed) Hand- Phycol 21:509–517
book of microalgal mass cultures. Blackwell Science, Oxford, 85. Wiesner C, Kopecký J, Pflueger M, Hundsberger H, Entler B,
pp 485–512 Kleber C, Atzler J, Hrouzek P, Štys D, Lukešová A, Schuett W,
69. Strasser RJ, Srivastava A, Govindjee (1995) Polyphasic chloro- Lucas R (2007) Endothelial cell-based methods for the detection
phyll a fluorescence transient in plants and cyanobacteria. Pho- of cyanobacterial anti-inflammatory and wound healing promot-
tochem Photobiol 61:32–42 ing metabolites. Drug Metab Lett 1:254–260
70. Strasser RJ, Tsimili-Michael M, Srivastava A (2004) Analysis of 86. Xiong FS, Kopecky J, Nedbal L (1999) The occurrence of UV-B
the Chlorophyll a Fluorescence Transient. In: Papageorgiou absorbing mycosporine-like amino acids in freshwater and ter-
GC,Govindjee (eds) Chlorophyll a Fluorescence: A Signature of restrial microalgae (Chlorophyta). Aquat Bot 63:37–49
Photosynthesis. Advances in photosynthesis and respiration, vol 87. Zahradnı́k J (1967) Bioengineering. Mass culture of Scenedesmus
19. Springer, The Netherlands, pp 321–362 in outdoor units. In: Nečas J, Lhotský O (eds) Ann. Rep. Algolog
71. Šetlı́k I (1970) Analysis of photosynthetic activity in algal culture Lab. Třeboň for 1966, pp 103–125
under various climatic conditions. In: Dykyjová D (ed) Produc- 88. Zaťková I, Sergejevová M, Urban J, Vachta R, Štys D, Masojı́dek
tivity of terrestrial ecosystems—production processes. PT-PP J (2010) Carotenoid-enriched microalgal biomass as feed sup-
Report No. 1 (1964–1969). Czechoslovak Academy of Sciences, plement for freshwater ornamentals: albinic form of wels catfish
Czechoslovak National Commitee for the International Biological (Silurus glanis). Aquacult Nutr. doi:10.1111/j.1365-2095.2009.
Programme, Subcommittee for PT-PP/IBP, Praha, pp 167–174 00751.x
72. Šetlı́k I (1959) Perspektivy pestovania rias pre potravinárské a 89. Zelı́k P, Lukešová A, Voloshko L, Štys D, Kopecký J (2009)
krmivárské účely. Naša veda 6:114–118 Screening for acetylcholinesterase inhibitory activity in cyano-
73. Šetlı́k I, Komárek J, Prokeš B (1967) Short account of the activ- bacteria of the genus Nostoc. J Enzyme Inhib Med Chem
ities from 1960 to 1965 and some future prospects. In: Nečas J, 24:531–536
Lhotský O (eds) Ann. Rep. Algol Lab. Třeboň for 1966, pp 7–38 90. Zelı́k P, Lukešová A, Voloshko LN, Štys J, Kopecký J (2010)
74. Tamiya H (1955) Growing chlorella for food and feed. Third Nostotrebin 6, a bis(cyclopentenedione) with cholinesterase
World Symposium on Applied Solar Energy, Arizona, inhibitory activity isolated from Nostoc sp. str. Lukešová 27/97.
pp 231–241 J Enz Inhib Med Chem 25:414–420
75. Tamiya H, Hase E, Shibata K, Mituya A, Iwamura T, Nihei T, 91. Zhu XG, Long SP, Ort DR (2008) What is the maximum effi-
Sasa T (1953) Kinetics of growth of Chlorella with special ref- ciency with which photosynthesis can convert solar energy into
erence to its dependence on quantity of available light and on biomass? Curr Opin Biotechnol 19:153–159
temperature. In: Burlew JS (ed) Algal culture: from laboratory to
pilot plant. Kirby Lithographic, Washington, DC, pp 204–232

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