Eng. Life Sci. 2014, 14, 560573 www.els-journal.

com

Mark Fresewinkel1 Review

Rosa Rosello1
Christian Wilhelm2
Olaf Kruse3
Ben Hankamer4
Clemens Posten1
1
Institute of Process Engineering
in Life Sciences, Section III
Bioprocess Engineering,
Karlsruhe Institute of
Technology, Karlsruhe, Germany
2
Department of Plant Physiology,
Institute of Biology I, University
of Leipzig, Leipzig, Germany
3
Algae Biotechnology and
Bioenergy Group, Department of
Biology, Center for
Biotechnology, Bielefeld
University, Bielefeld, Germany
4
Institute for Molecular
Bioscience, The University of
Queensland, St Lucia,
Queensland, Australia
Integration in microalgal bioprocess
development: Design of efficient, sustainable,
and economic processes
The use of microalgae as a production system has gained huge interest in recent years.
Recent research has concentrated on single aspects, such as the microalgal cells or the
photobioreactors. The design of sustainable, effective, and economic processes for
microalgal products requires the integration of microalgal biology including strain
selection and genetic engineering, process, and reactor design and integration into
environmental mass and energy fluxes. This involves tools of biotechnology and pro-
cess engineering. Several attempts of such integrated processes have been developed.
This review groups the integration aspects into several degrees of integration with
respect to the classical upstream, bioreaction, and downstream steps. The integration
levels consider metabolic, process, environmental, and social integration. At the end,
the following question remains: How far can microalgal biotechnology help to solve
the problems of feed, food, and fuel supply and contribute to a better society? Over-
all, this review gives an overview of the different stages of integration in microalgal
processes. It is a guide towards conceptual positioning and planning of microalgal
plants. Furthermore, it is a decision guide to support microalgal technologies.
Keywords: Biorefinery / Cross-feeding / In situ product removal / Process integration / Strain
selection
Received: March 7, 2014; revised: June 6, 2014; accepted: June 30, 2014

DOI: 10.1002/elsc.201300153

1 Introduction To move from the biological potential to a sustainable and

The broad range of microalgal products fascinates scientists
worldwide [15]. The products include valuable oils, proteins,
and carbohydrates as well as pigments, vitamins, and minerals.
The concern about the future welfare of our world initiated the
discussion on the contribution of microalgae to fuel, food, and
feed for a rapidly growing population [6]. Anticipated applica-
tions are found in high-value products such as pharmaceuticals
or neutraceuticals, in fine chemicals and basic food supply, and
finally in bulk chemicals and fuels. While algae contribute to
high-value products due to their unique abilities to form com-
plex molecules, the composition of the cells qualifies them to be
a strong link in the food chain. Only the application as low-value
bulk products or fuels can lead to a remarkable substitution of
fossil oil and potentially to a reduction of global warming.
Correspondence: Dr. Clemens Posten (clemens.posten@kit.edu),
Institute of Process Engineering in Life Sciences, Section III Biopro-
cess Engineering, Karlsruhe Institute of Technology, Fritz-Haber-
Weg 2, 76131 Karlsruhe, Germany
Abbreviations: FAAE, fatty acid alkyl esters; IR, infrared
economically viable production, the integration of microalgae
into processes, into the environment, and into our society is re-
quired. Process development comprises the selection of strains
and media in an upstream stage, the cultivation in photobioreac-
tors in a bioreaction stage, and product recovery and formulation
in the downstream stage. In this direction a number of accom-
plishments have already been made, but more work is waiting to
be done. In addition to the optimization of single-process stages,
the structure of entire processes has to be reviewed. Besides prob-
lems of process stability in outdoor production units, the chal-
lenge of an energy and CO2 -neutral production and consump-
tion cycle has to be met. Behind open technological questions
even more problems of integration of microalgal biotechnology
into our society with market constraints and cultural realities are
lurking.
Many small- and middle-sized companies have been founded
but disappeared after the first failure either of their technology
or their financial framework. But other enterprises appear to be
having better success than their predecessors. Also in politics
algae technology is supported. In Europe a new regulation aims
at substituting 10% of the fuel consumption by biofuels of the
second generation, preferably from algae and agricultural wastes

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(European Commission, Renewable Energy Targets by 2020).
Solutions are not found in single technologies but in interplay
of different fields of biology and engineering, as well as social
and cultural sciences. Economically viable applications depend
on local markets and the availability of human resources. Fur-
ther anticipated developments with respect to the mentioned
integration aspects are outlined in this review.
2 Integration on the level of the standard
process structure
The development of bioprocesses generally can be divided into
three stages, as described in Fig. 1. It consists of the upstream
process in which potential organisms are screened and geneti-
cally engineered to suit process targets such as productivity and
product titer. Based on these strains a process is developed to
meet the specific demands of the cells and to reach a high yield
of the product. Impacts of engineering are the reactor design, the
applied media as well as process parameters such as pH and tem-
perature. The third step of the process development addresses
the recovery of the product. Depending on the product and its
occurrence this may require several process steps such as cell
separation, disruption, and purification. Even though the mate-
rial flux is going from upstream to downstream, the design of
efficient bioprocesses requires an extensive review of the entire
process already in the development stage. Information of each
process step needs to be considered to evaluate the effects of one
process part to the other. Different disciplines of microbiology,
process engineering, analytics, and monitoring and control tech-
niques come together. This can be regarded as the integration of
the scientific disciplines.

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Figure 1. The material and infor-
mation flux of a bioprocess de-
velopment. In the stages of the
bioprocess development, differ-
ent engineering and natural sci-
ence disciplines have key activi-
ties and have to cooperate.
2.1 Cell properties support overall process
performance
Algae are found in a variety of different environments such as
marine and freshwater as well as different terrestrial habitats.
They have a broad phylogenetic diversity, resulting in a broad
range of metabolic and biochemical properties. The number of
algal species has been estimated at between 200 000 and 1020
million species [7, 8]. Most of them are microalgae. In addition,
a few of these strains can be further optimized by genetic en-
gineering [9]. Despite this abundance of microalgae and their
possible applications, only few of them are used in biotechnol-
ogy. In the last years strains have been selected with high yields of
high-value products [1] but also of oil [10] or starch [11]. The di-
versity can be further exploited by seeking for cells that are more
streamlined to specific process conditions, as outlined in Fig. 2.
Concerning a contribution of upstream operations to the
performance of the bioreaction and downstreaming, the focus
could be more on process-oriented strain selection. By consider-
ing process relevant parameters in the choice of the algae strain,
the effectiveness of the whole process can be increased. This
direction could be titled as increasing cell robustness. Next to
specific growth rate and high product titers, screenings for a
process-oriented strain should consider mechanical properties
of the microalgae. Increased robustness of the algae against me-
chanical shear stress caused by pumps and bubbles may lead to
higher growth rates and cell concentrations [12, 13]. Addition-
ally, the robustness of the process concerning physical parameters
of the bioreaction has to be broadened. A high growth rate and
profitable composition of the algae over a broad range of tem-
perature is desirable to decrease the required energy input for
temperature control [14]. For the required light intensity, two
cultivation strategies have been developed in the recent years.
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The first approaches the achievement of high growth rates at
low light intensities as in case of high cell densities or light di-
lution [15]. In the second strategy the saturation effects of high
light intensities are considered. It has been hypothesized that a
reduction of the antenna pigments, the antenna truncation, al-
lows deeper permeation of light into the reactor with increased
growth rates at high light intensities by reducing nonphotochem-
ical quenching [16] on the illuminated side of the reactor. In this
case no or less light dilution on the level of reactor geometry
would be necessary. This would allow higher productivities for
constant high biomass concentrations, for example under con-
tinuous cultivation. However, until now no higher productivity
under natural light climate has been shown.
In case of open ponds, tolerance against bacteria, predators,
and pesticides is a further aspect [17]. Also here strategies have
to be found. Even seemingly simple things such as medium com-
position have to be revisited [18]. Many groups in research and
industry work on renewed recipes to allow higher cell densities,
avoiding organic buffers (cut of cost and contamination) and
providing a completely balanced nutrient composition for easy
medium recycling [19]. By balancing the medium composition
the biomass can be customized for a desired product, to increase
yield and productivity in terms of biomass, starch, or lipids [20].
While in nature many algae strains prefer to be fixed on solid
supports, sticking to walls and flocculation is an unwanted be-
havior of cells in classical suspension cultures. That could be
prevented by looking at different strains during the screening
phase by choosing for example habitats with freely suspended
cell populations with a suitable cell-surface structure.
Smart strain selection can also make downstream operations
easier. In case of intracellular products conventionally, the down-
stream process requires a solidliquid separation step to concen-
trate the biomass, for example by filtration or centrifugation [21].
These processes are energy- and cost-intensive due to the small
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Figure 2. Screening criteria for
algae in integrated bioprocesses
need to address demands of the
cultivation, the process stability
as well as the downstream abili-
ties of the desired product.
particle sizes in the range of a few micrometers, a density close to
the surrounding medium and a negative charge of the microor-
ganisms that prevent them from flocculation [21]. For higher
process efficiencies, flocculation on demand can be induced in
algae by increasing the pH of the medium due to carbon dioxide
consumption [22]. Therefore, the carbon dioxide supply sim-
ply has to be reduced at the end of the cultivation. Also using
filamentous algae is an option to reduce efforts in the down-
stream process [23, 24]. Further downstream process steps such
as extraction or cell disruption can be optimized by considering
the cell wall of the production strain already during the strain
selection phase.
An alternative type of cultivation is the growth of algae in
biofilms. This approach is less studied than suspended cultures
but could reduce auxiliary energy input and cell harvesting costs
due to higher cell densities. It has already been shown to be
advantageous for diatoms and benthic algae [25, 26]. Like many
other microorganisms, they tend to live attached to surfaces.
Instead of forcing them into suspension, cultivation on plastic
sheets or submersed strings allows easy harvesting by shedding.
Material studies for biofilm supports show areal biomass pro-
ductivities of up to 2.6 g/(m  d) dry weight of Chlorella sp. on
a simple sheet of polystyrene foam [27] or 50 . . . 80 g/(m d)
on a cellulose filter [28]. More research is currently done in this
field [29, 30]. In case of extracellular products [31] or in appli-
cation of wastewater treatment [32] the cells can remain on the
surface. For the recovery of intracellular products or biomass
the cells can, for example, be harvested by scratching off the
surface [33, 34]. The solids content of the harvested biomass
is in the range of values after centrifugation [34]. On the sur-
face remaining algae are the new inoculum of the next growth
cycle [27].
Bassi has called the process of finding and developing suit-
able strains for production as domestication of the algae [35].

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It lasted millennia until grains developed from grass only by
selection. Practical optimization of yeasts strains for bread and
beer production (e.g. ethanol tolerance, flocculation properties)
was done intensively since more than a century. For algae, the
process of getting strains well adapted to the specific situation
in the reactor is expected to last not even one decade. While in
research antenna truncation is reached by genetic engineering,
the better understanding of the benefits could finally results in
looking for appropriate strains in nature, especially as most algae
are able to adapt their antenna size to light conditions. So the di-
versity could be exploited straighter. On the middle term, genetic
engineering cannot be avoided for further progress. Targets for
genetic engineering are sharper product profiles, improved light
performance or pesticide resistance. Genetic engineering will be
applied to microalga as a normal tool like it is already done in
industrial biotechnology and in parts of agriculture despite the
untapped natural diversity.
2.2 Cultivationthe centerpiece of the process
Recent efforts have been done to increase achievable productiv-
ities by developing new designs for photobioreactors [3639].
Targets are a better light distribution on the reactor surface, a
better light dilution into the volume, and a minimized energy
input for aeration and mixing. The basic challenge in reactor de-
sign is to transform the given conditions outside the reactor with
respect to light, CO2 -availability, and heat into conditions inside
the reactor preferred by the algae given as qualitative knowledge
or quantitative kinetics. Here integration between biology and
process engineering science is necessary. Not only overall spe-
cific growth rates but also macromolecular composition, stress
markers for example for high oxygen concentration or high tem-
perature as well as morphological appearance are signals of the
cells to tell about its reaction to the specific conditions in the
reactor, as described in Fig. 3. The interpretation of these signals
could trigger the next step forward in rational reactor design.
Reactors differ with respect to product value and environ-
mental condition such as light intensity. While tubular reactors

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Figure 3. Basic idea for reac-
tor design; the reactors trans-
form environmental conditions
to physiological conditions, a
process described and designed
by engineers in terms of en-
ergy and mass flows from the
environment into the reactor
and reaction rates governed by
kinetics and stoichiometry of
the cells. Reactor development
should take more notice from
cell reactions caused by the spe-
cific situation inside the reactor.
have proven their ability to be scalable and exhibit high produc-
tivity, plate reactors have the advantages in lower specific energy
input. Artificial illumination by LEDs is good for research and
very high-value products. So there will be no single design for all
purposes in the foreseeable future. Inside every design pattern a
reliable parameterization is demanded.
There are two persistent specific problems only indirectly re-
lated to reactor design. The first one is overheating. Even in
Middle Europe, outdoor reactors have to be cooled by water
spraying. Ideas for a sustainable temperature control are cov-
ering the reactors with infrared (IR) reflecting material, using
the thermic inertia of the ground by installing heat pumps or
employing phase-changing polymers. The next genuine chal-
lenge in outdoor cultivations is fighting against contamination.
In the harmless case, a selected species for example with high
oil content but low growth rate is permanently outcompeted by
wild strains that grow faster without the metabolic burden of
product formation. It is not expected, that the so-called closed
bioreactors are really closed or allow for an axenic operation
in large scale of hectares or square kilometers. Here probably
only a techno-biological approach will be helpful, the choice of
extremophile microalgae being only the first step. The use of
herbicides with genetically engineered herbicide resistant algae
is reality [40]. However, microalgae technology thus loses its
flawlessness and makes integration into the society more diffi-
cult. In addition more practical aspects have to be done, such as
minimization of the amount of plastics, choosing recyclable ma-
terials, and care for robustness against bad weather conditions
or animal attacks. These examples show that the integration of
reactors to fulfill the biological needs on the one hand and the
needs to fit into a real existing environment on the other hand is
not fully implemented.
In addition to the design of the reactors and choosing pro-
cess operation strategies, the measuring and control techniques
also have to be adapted to algae-specific demands. This is espe-
cially true as in outdoor production facilities highly fluctuating
light intensities and temperatures are faced. Supplying carbon
dioxide according to the light-depending photosynthetic activ-
ity is an option toward lower energy consumption and higher
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productivities. The regulation of mixing energy and carbon diox-
ide input in dependence of biomass concentration and activity
as well as the radiation of light can contribute to the reduc-
tion of auxiliary energy [41]. This approach is often claimed
but rarely implemented. For the realization of the above point,
optical measuring techniques can be applied due to clear media
and specific optical characteristics of the cells. Light-scattering
techniques give information about morphological parameters of
the cell [42] and fluorescence measurements such as pulse am-
plitude modulation allow the determination of the physiological
activity [43]. Due to the effects of the carbonate system on the
pH, limitation in carbon can be avoided via simple measurement
of pH and regulation of the CO2 content in the aeration [44].
Final biomass concentration can be further increased by feed-
ing strategies for example for ammonia or phosphate. These
compounds cannot be in the fresh medium in sufficiently high
concentrations.
2.3 Downstreamnot a standalone approach at the
end of the pipe
Solidliquid separation in bioprocesses, which means harvesting
on the functional level, has been optimized since decades, result-
ing in standard application of disk stack centrifugation and vac-
uum drum filtration potentially coupled with flocculation and
filter aid usage. From a stand-alone engineering view that should
be applicable for algae cultivation as well. However, there are dif-
ferences in the cultivation parameters and the targeted markets,
being a serious integrated aspect in cell separation of microal-
gae. Depending on the reactor system, biodrymass concentration
ranges between 1.5 g/L for open-pond systems and 3.5 g/L for
closed reactor systems. Even 10 g/L are possible with appropriate
feeding strategies and short light pathways, even though last has
not been transferred to pilot scale jet [45]. On the other hand,
the anticipated usage as sustainable biomass for fuels makes a
possible market value lower comparing to other biotechnologi-
cally produced bulk products. Another biological aspect coming
to this unit operation is that algae containing considerably high-
oil contents have the same or even a lower density than water
and are therefore not accessible by centrifugal forces. Altogether,
that leads to much more rigid constraints with respect to costs
and energy consumption. This situation had to be envisaged by
innovative separator designs and modes of operation [46].
But still harvesting is one of the most energy- and money-
consuming steps. The burden cannot be laid solely on the isolated
view to this unit operation. For microalgae this means that an
increasing cell density can be paid back by a more efficient sepa-
ration step. To double the cell concentration from, for example,
3 to 6 g/L means reducing the water flux through the centrifuge
and therefore the energy demand for harvesting by 50% per
kilogram algae dry mass. But higher biomass concentrations in
the bioreactor can lead to steeper light gradients or larger dark
zones in nonoptimized reactors. Respiration increases with the
result of a relatively lower productivity. This can only be com-
pensated by faster mixing, of course, on cost of auxiliary energy
consumption. On top of it, assuming even a constant productiv-
ity with constant light availability, cell age and therefore product
quality may change with changing dilution rate. These complex
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Figure 4. Productivity and product concentration in continuous
microalgal cultivations, adapted from [51]. Here the window of
operation is defined by an optimal choice of the countervailing
courses of biomass concentration and productivity.
interactions between several process steps with different chemi-
cal, physical, and biological features and engineering constraints
can be handled only with model-based optimization tools. In
chemical industry, models for the prediction and optimization
of the process are standard and simulation software has been
used for many years and already adapted to biotechnological
processes [47, 48]. A graphical analysis to evaluate the influence
of significant process interactions has been applied by the win-
dow of operation [49,50]. This tool enables the process designer
to visualize the results of a flow-sheet simulation and helps to
increase the process performance by improving the design and
parameterization of the process. To apply this tool, target pa-
rameters such as a specific level of product recovery and cell
debris removal are determined, as in the heterotrophic exam-
ple of [49]. Significant process interactions and their influence
on subsequent unit operations are considered. In 2D maps, the
area in which the target parameters are achieved is marked. The
dimensions of the map are given by significant influence param-
eters of different process steps. The concept of window of oper-
ations helps finding for example an optimum operation point
with respect to the cell concentrations and productivity [51], as
shown in Fig. 4.
Downstream processing is often regarded as a stand-alone ap-
proach at the end of the value-added chain. This is not entirely
true, as already shown in the paragraphs concerning screen-
ing. Downstream processing is or should be additionally directly
affected by process parameters of the cultivation. These interac-
tions between the single steps, as described in Fig. 5, have to be
considered and adapted.
But there are more interactions between harvesting and cul-
tivation. In contrast to other microorganisms, microalgae pass
through a diurnal cell cycle synchronized by the solar radia-
tion. Over the day, storage components are accumulated that are
respired and metabolized to proteins and other cell compart-
ments during the night [52, 53]. If enough light is provided over
the day, the cell division is also synchronized to the daily cycle.
These cycles have to be considered for the choice of continuous

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or semicontinuous processes and for cell-harvesting cycles. As-
suming a constant productivity, harvesting for example 50% of
the suspension every day leads to cell doubling times of 1 day
but at low biomass concentrations while a lower daily harvesting
portion results in lower specific growth rates but at higher cell
densities. As that changes the average cell age as well, repercus-
sions on the product quality can be assumed.
Integration of process steps does not only concern the extrac-
tion of extracellular and intracellular products. Further treat-
ment steps after the product removal might be necessary. In
case of biodiesel production (fatty acid alkyl esters, FAAE), the
conventional route involves conversion of the extracted lipids to
FAAE and glycerol [54,55]. The integration of the extraction and
conversion step of the microalgae lipids has recently been facil-
itated by Ehimen [56]. They investigated the in situ transester-
ification process of FAAE directly from the oil-bearing biomass
by using a homogenous acid catalyst. This process eliminates
the solvent extraction step required to obtain the oil feedstock
as in the conventional method. These two aspects have shown
that harvesting is not only an end of pipe technology but can be
positively influenced by a suitable concept in the upstream and
the cultivation stage and has serious effects on product quality
and product costs.
3 Integration of single-process steps into
each othera way to process
intensification
In addition to the optimization of single-process steps, the entire
process structure has to be also reviewed to attain high overall
process efficiencies. That can mean to implement for example
two stages or phases instead of one, in cases where two consec-
utive different conditions perform better than one process step.
An example for cultivation is the decoupling of growth phase

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Figure 5. Reduction of process
steps due to integration; some
technical steps can be simplified
or even omitted by letting the
algae cells do their job.
and product-formation phase for example for oil or carotenoid
production. In solidliquid separation, a preconcentration step
for example thickening by a decanter may precede the concen-
tration by a disc stack centrifuge. In other cases, process steps
are shifted toward and into each other to gain synergisms. This
has been shown already above for downstream processing. In
simple cases, it saves transport ways between the cultivation and
the separation unit or positively influences process kinetics. The
highest level of integration can be succeeded by allowing the cell
to do what it can do best. This means not only to synthesize
a product but performing further refinement and product iso-
lation by itself, saving as much technical steps and devices as
possible.
3.1 Integration of cultivation and downstream
in situ product recovery
An intensive process operation is achieved by the integration of
single-process steps into each other, forming one step. This is the
case for in situ product removal. The combination of product
recovery and cultivation can save the cell-harvesting step and
can lead to a continuous product stream. For the cultivation
this means operation in a steady-state modus, avoiding cellular
energy expenditure for cell mass formation. In the best case
the product is nonwater miscible, volatile, or gaseous, making
additional extraction or separation steps easy or superfluous, as
described in the further processes.
Polysaccharides are the most common extracellular products
from microalgae, which allows in principle for the employment
of in situ product removal. While agar-agar or carageenan
from macroalgae harvested in more or less natural environments
have already been brought to the market, polysaccharides from
microalgae are less commonly produced. They are proposed
for high-value applications [57] but less for food, chemicals,
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or fuels. Obstacles are the relatively low calorific value and the
problems during separation [58], which is not much easier than
cell separation. New and more specific approaches may change
that in the future.
However, spontaneous excretion of metabolites with higher
value or energy content is quite seldom, with the colonial green
algae Botryococcus being a rare example. This hydrocarbon ex-
creting alga has been intensively investigated. A possible extrac-
tion of the hydrocarbons stored in the outer matrix of Botryococ-
cus braunii cell colonies has been shown by [59, 60].
Stimulus of product excretion without affecting the algae too
much is known for example by the addition of specific solubles
or by applying stress on the algae. An example of a soluble-
induced excretion is anthraquinone from C. glabra [61]. The
excretion of hydrogen can be induced in C. reinhardtii upon
sulfur deprivation [62,63]. In this case the product H2 is formed
quite close to photosynthesis on the metabolic level without
requiring adenosine triphosphate dependent synthesis steps, it
is directly marketable and leaves the reactor without any extra
energy expenditure for separation due to its volatile character.
Of course, the problems lie in the details such as removing the
oxygen. Despite these advantages, current problems with low
energetic efficiency have prevented a further scale up until now.
To come to intracellular products by in situ separation harsher
conditions are required. One well-known example that has been
applied to microalgae is the extraction of products by direct con-
tact of a solvent with the growing biomass [64]. This milking
has been done with a two-phase system with water-immiscible
solvents to extract carotenoids from Dunaliella [6567]. The in-
teraction of the solvent and the biomass can cause permeabiliza-
tion of the cell membrane or even lead to cell disruption, which
limits this approach. Cloud-point extraction of hydrophobic
products with surfactants is an alternative to the solvent-based
extraction [68].
3.2 Integration on the cell levelthe cell factory
While the excretion and consecutive in situ solidliquid separa-
tion or extraction are an option in some specific cases, some ef-
fort is made to control product excretion by genetic engineering.
Background to this way of thinking is the idea that cells can do
some things better than chemical plants. This means primarily to
form enantiopure products, performing regiospecific substitu-
tions on complex molecules, and synthesize sequentially ordered
polymers. But the cell can do more, namely to build up spatially
ordered structures including transportation of molecules to the
place inside or outside the cell where they belong. The underlying
mechanisms have already been discovered for the case of pro-
tein transport into different cell compartments (Blobel, Nobel
prize 1999). So the cells are an excellent highly specific separator
machine. This concept has been published under the label cell
factory understanding that the most macroscopic steps could
be made by the cell itself behaving like a microscopic factory.
The technical process only has to give a favorable environment.
The advantages of photo-bioprocesses for the production of
extracellular recombinant proteins have been shown for moss
cells by Physcomitrella patens [69]. These are among others
the reduction of danger of viral contamination, clear mineral
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medium for better solidliquid separation, and potentially a
suitable state of glycosylation. The integration of solidliquid
separation steps for cell recycle and protein separation, for ex-
ample cross-flow filtration [70] or magnetic separation [71, 72],
is therefore possible and can remarkably reduce the number of
more complex downstream processes such as chromatography.
Phototrophic production of extracellular proteins is still feasi-
ble only for high-value products. For microalgal processes, these
approaches started later with Chlorella [73] due to the more com-
plex genetics. But recently more and more promising examples
are published targeting not only the pharmaceutical high-price
segment [74, 75] but also middle-price products for the food
industry [76].
Not only for proteins but also for the extracellular production
of smaller target molecules, phototrophic cells can be designed
by genetic engineering. This step is necessary to reach for the goal
of phototrophic production of transportation fuels. Algal cells
engineered by synthetic genomics could be designed to secrete
oil through their cell walls for an increased biodiesel production
(www.syntheticgenomics.com/what/renewablefuels.html). On a
328 m site, Synthetic Genomics intends to test and scale up
engineered strains of algae.
A similar process integration strategy is designed by the cor-
poration Joule (Bedford, MA, USA). For the production of diesel
a cyanobacterial organism is designed to synthetize alkanes that
do not require further processing. The organism is engineered to
switch between a growth and a production phase. An additional
secretion system releases the product directly into the surround-
ing medium. The medium is circulated through a separator to
extract the product. Three days of algae growth are followed by
53 days of continuous production with no more biomass in-
crease [77]. In this production phase inorganic CO2 is converted
to hydrocarbon products instead of biomass. This platform can
be tailored to multiple products by applying different engineered
organisms. In the focus are ethanol and hydrocarbons for diesel,
jet fuel or gasoline. In this process the loss in photon conversion
efficiency from photon to final product is low due to a reduction
in process steps and the losses in biomass buildup.
A next logical step is to change the natural product to an-
other one like ethanol by inserting an additional pathway into
the algal cell. Here the advantage is envisaged that ethanol
leaves the cell simply by diffusion. Algenol Biofuels (Bonita
Springs, FL, USA) developed an ethanol process in which a trans-
genic alga directly converts sunlight energy and carbon diox-
ide into ethanol (http://www.algenolbiofuels.com/direct-to-
ethanol/direct-to-ethanol). Furthermore, the produced ethanol
is directly evaporated by the heat of sun radiation. This allows
a continuous process without cell separation. Ethanol is a mar-
ketable product. Thereby chemical processing can be avoided and
integration into the market is potentially easy. Whether the an-
ticipated areal productivities usually expected for biomass from
a designed plant of several hectares in Mexico can be reached
is not proven, but this process demonstrates the possibility of a
highly integrated process design.
To benefit from the photosynthetic capacity of the algae, the
intention should be the autotrophic usage of the cell. To attain
the desired final product, one specific microalga may not be the
best producer. In the case of ethanol product inhibition could
be a problem, as algae are not ethanol resistant by nature. In

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www.els-journal.com Eng. Life Sci. 2014, 14, 560573
such cases metabolic steps of two different microorganisms can
be integrated into one process. A primary organism converts
the primary substrate, in case of microalgae light and carbon
dioxide, to a primary product. This primary product serves as
a substrate for a following heterotrophic culture. Primary prod-
ucts as intracellular starch, extracellular polysaccharides, or even
the cell wall can be provided to the subsequent heterotrophic
steps, for example ethanol [78]. This process of cross-feeding is
already known from natural symbioses between bacteria and al-
gae. Vitamin B12 is acquired to algae by a relationship to bacteria,
while the algae supply carbohydrates in return [79]. The supply
of carbon dioxide as well as the reduction of photosynthetic
oxygen tension in the microenvironment increases the growth
rate of microalgae [80,81]. Nevertheless co-culturing within one
reactor is limited by mutual shading of algae by bacteria as well
as optimum values for the respective environmental parameters.
That can require different cultivation compartments.
An example for a technical realization is the con-
version of microalgae produced starch into ethanol
(http://www.oilgae.com/algae/pro/eth/eth.html). Microal-
gae with high-starch contents are hydrolyzed by extracellular
amylases of Aspergillus and finally converted to bioethanol by
yeast, analog to the conversion of starch from corn. The ethanol
can be used as a fuel or as a platform chemical. Another example
is the production of isoprene [82] or ethanol by SolixBioSystems
(Fort Collins, CO, USA) and Glycos Biotechnologies, Inc.
(Houston, TX, USA) from microalgae-excreted oils.
Microalgae are discussed as perfect photosynthetic organ-
isms on the one hand, and as producers of high-value com-
pounds on the other. Both functions can well be separated in

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
Figure 6. A possible pro-
cess/reactor structure for the
realization of the concept
of new green chemistry.
Shown is the example of
photo-methane, adapted
from [31].
principle in the sense of cross-feeding. The company Solarzyme
(San Francisco, CA, USA) produces high-value lipids by het-
erotrophic grown algae on a substrate originating from sugar
cane (www.solazyme.com/market-areas). This uses the ability of
the algae to accumulate high-oil concentrations. But as the al-
gae are fed with sugar, the whole process is only as energetically
effective as the production of sugar cane. Here a first stage with
another microalga producing carbohydrates as feed could po-
tentially bring more energetic efficiency to the process. A similar
process is performed by Flaschl (Bielefeld, Germany), using Eu-
glena to produce Paramylon, a -(1,3)-glucan presumed to have
immunostimulating effects [83, 84].
3.3 A concept of high-level integrationnew green
chemistry
In general, photosynthetic products should be used as the sub-
strate for cross-feeding, which require only little metabolic en-
ergy during its production in the cell. This principal has been
implemented in a process, where a Chlamydomonas biofilm pro-
duces and excretes only glycolate, an early metabolite of the
photorespiration [31] (Fig. 6). The excretion can be induced by
photorespiratory conditions, that is, a high-oxygen partial pres-
sure. Further enhancement is envisaged due to deletion of the en-
zyme glycolatedehydrogenase to suppress further metabolism of
glycolate. The produced glycolate diffuses through a membrane
into a second compartment to be degraded to biogas by anaer-
obic bacteria. The last step is a realization of a gaseous product
leaving the reactor without any further mechanical solidliquid
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separation steps. Alternatively to biogas, the production of other
compounds can be foreseen. In this way the idea of cross feeding
and in situ product removal is combined in a continuous pro-
cess. Furthermore only low amounts of energy are wasted in the
growth of algae and highly valuable metabolites or in mechanical
separation steps.
Employment of biofilms has been mentioned above as a
means of reducing energy for downstream processing with re-
spect to filtration or centrifugation. Combining this idea with
the idea of product excretion and in situ separation brings an
additional quality into microalgal production processes. On the
level of cultivation, mixing is no longer necessary in the usual
amount. No high specific growth rates are required but rather
the cells are allowed to use metabolic energy only up to an extent
necessary for maintenance purposes, for example DNA repair or
protein turnover. In this way, metabolic energy can be channeled
to the excreted product. The excreted product can consequently
be used to feed other microorganisms to get the final product.
This concept has recently been published under the key term
new green chemistry [82].
The concept of new green chemistry comprises a conse-
quent minimization of energy by restricting transport to diffu-
sion of educts and products, minimizing metabolic energy by
employing biofilms close to zero growth, and aiming at a prod-
uct being formed in the central metabolism and finally construct
a robust and cheap design. Future practical implementation will
show whether this photosynthesis-driven technology will be a
feasible option for energy production with nearly zero CO2 -
emission. At least it can bring microalgal biotechnology to large
scale with the anticipated high yields and low costs to support
remarkably the bioeconomy of the future.
4 Embedding microalgal biotechnology into
ecology, economy, and society
4.1 Integration into existing environmental energy
and mass fluxes
Already existing examples for sustainable processes using mi-
croalgae are based on the smart interconnection of existing en-
vironmental mass and energy fluxes. On chemical sites, this
principle has already been implemented. Efficient use of re-
sources is realized by networking several production plants.
This interconnection creates value chains in which byprod-
ucts of one plant can be used as an educts of other pro-
cesses. In this way, processes are designed exhibiting re-
duced energy, emissions, and cost efforts (BASF, Ludwigshafen,
Germany) (http://www.basf.com/group/corporate/en/investor-
relations/basf-in-brief/verbund/index). Applied to microalgal
cultivation, logistical and financial challenges include the water
and nutrient demand of the algae as well as auxiliary energies.
Economic processes have already been developed basing on the
smart interaction with existing environmental mass and energy
fluxes, as described in Fig. 7.
Supply of seawater to compensate evaporation is the most
obvious solution for the water-management problem. Near-
costal grounds are available for algae plants in square kilo-
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Figure 7. Microalgae have the advantage to be used as whole
cells or after decomposition of different molecular fractions; the
structure of the biorefinery changes with changing product value
and the market.
meters scale using seawater. Also, algae cultivation with swim-
ming photobioreactors is in the range of things that are con-
sidered (http://www.nasa.gov/centers/ames/research/OMEGA).
But also open ponds in the middle of a continent using brackish
ground water are described (Cellana Inc., San Diego, CA, USA).
In this context the usage of cheap industrial low-temperature
heat for temperature control of closed photobioreactors should
also be mentioned. Locations in the middle latitudes that are
sunny in early spring but too cold for algal cultivation could be
opened for operation by employing that option.
Next to water supplies, nutrients could become a logistic,
ecological, and financial problem. While ammonia production
is an energy-efficient process, phosphate is anticipated to become
scarce in the future. Of course, it is a must to recycle nutrients and
water directly at the microalgal production plant wherever pos-
sible [19, 85]. After extraction of oil and using proteins for feed,
phosphate could be recovered from the residual biomass. On the
other hand municipal and industrial waste water as dairy ma-
nure effluent is a possible nitrogen and phosphate source for the
algae [33, 86]. Next to lower process costs of the microorganism
cultivation, a waste water treatment is achieved. Consequently,
a lot of research in this direction has started in the last few years.
The goal is to install a shortcut between buying expensive ni-
trogen compounds as proteins or fertilizers on the market and
degrading them finally in the waste water plant. Here algae can
make a valuable contribution, not only to sewage treatment but
also to recycle valuable nutrients.
Flue gas from coal or gas combustion, off-gas streams from
chemical plants or lime kilns as well as other technically avail-
able carbon dioxide emitters are often proposed as CO2 -source.
However, usually there is by far not enough land around in-
dustrial plants to process all the off-gas and possible microalgal
production sites are located remote from industrial areas. The
deeper meaning of using flue gas does not lie in cleaning the
gas but in supplying higher carbon dioxide concentrations to
the algae suspension. That is necessary to overcome the mass
transfer limitations between gas phase and fluid phase and to
elevate the dissolved carbon level to avoid carbon dioxide uptake

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www.els-journal.com Eng. Life Sci. 2014, 14, 560573
limitations. CO2 -limitations can be avoided by controlling the
pH of the medium via the partial pressure in the aeration [87],
as described above in Section 2.2. Problems of SO2 -content with
respect to toxicity and pH effects and NOx -content as possible
nitrogen source are discussed in [88]. Coupling of algal culti-
vations to off-gas from heterotrophic fermentations as during
cross-feeding would allow for a partial carbon recycling directly
on a production site. Carbon dioxide from air would, in prin-
ciple, avoid all logistic problems. Here some work is going on
to investigate the thermodynamic limitations of carbon dioxide
concentration from the atmosphere.
The next necessity after water, nutrients, and carbon dioxide is
the demand for auxiliary energy. The installation of photovoltaic
panels on 25% of an algal cultivation area to produce electrical
energy has already been proposed without decreasing productiv-
ity [89]. That sounds at the first glance contradictory to the idea
of sustainability of algae, but it makes sense in so far as auxiliary
energy for the algae is mainly needed during sunny hours. Pho-
tovoltaic directly at the site can meet this demand better than
other energy sources as it makes conduction and storage of the
electrical energy nearly unnecessary. Furthermore, noneffective
wavelengths contained in the solar radiation can be used. IR ra-
diation cannot be converted by the photosystem of microalgae,
but causes heating of the cultivation medium. Instead of only re-
flecting IR radiation to reduce overheating, it could be converted
into electrical energy employing the so-called transparent pho-
tovoltaic panels. Even a modest efficiency of about 15% could be
enough to supply the energy demand of the peripheral devices
(pumps, pressured air, sensors). The final goal is the operation
of a stand-alone or plug-free microalgal plant.
4.2 Meeting demands of the markets by product
diversitythe biorefinery
For the present time, an energy neutral production of microalgal
biomass and therefore mass cultivation only for production of
chemical energy carriers is difficult. On the other hand, high-
value products are produced but in comparatively small amounts
covering only a few percentages of the cell dry weight. The first
idea is to use the residual biomass from the production of high-
and middle-value products for biofuel production [9092]. In
this way the whole cellall fixed carbon atoms so to speak
can contribute to additional value and the process as such does
not have to be energy-neutral. This idea of splitting the total
biomass into several products of different chemical composition
and value is called the biorefinery. The easiest way for a tech-
nical realization of this principle is to first extract the high- or
middle-value product and then secondly to convert the resid-
ual biomass in a biogas plant to methane. The energy from the
methane can be used for the plant itself or in some cases be
sold. Of course also nitrogen and phosphate from the effluent
of the biogas plant should be recycled closing mass and energy
cycles in the plant as good as possible. Other forms of biomass
usage such as hydrothermal conversion are subjects of current
research. Alternatively residual biomass can be used for aqua-
culture [93], presuming no solvent application in extraction was
employed and other prerequisites being given. But these attempts
have limited impact. Even if the number of high-value products

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www.biotecvisions.com
Figure 8. Stepping stone model to come to lower product prizes
and larger cultivation areas, experiences and earned money from
one level can drive the progress toward the next one.
will increase drastically, the total amount of saved energy and
carbon dioxide cannot contribute significantly to the solution of
worldwide problems.
But the situation can change. While microalgal technology
goes to lower costs and higher volumes, the biorefinery becomes
more and more meaningful (Fig. 8). In principle, proteins are
highly structured molecules, into which the cell has invested
metabolic energy. So they are far too valuable for burning and
should be used for food or feed. So one important stepping
stone will be to provide proteins for food and feed and building
blocks for technical polymers such as bioplastics [92]. Here the
biorefinery can also be more specific. Oil can be fractionated
into high-value polyunsaturated fatty acids as food supplements,
into middle-value chemical precursors, and into biodiesel using a
low-value fraction with saturated lipids. About 7% of the worlds
oil consumption is used for production of chemicals [94]. These
amounts are targets for being replaced by residual biomass from
higher plants and by oil from algae. Residual carbohydrates will
be used energetically as shown above. For these markets, it is not
even necessary to produce algae biomass with auxiliary energy
close to zero. This concept can indeed be of high value for food
supply and slightly reducing oil consumption, the prerequisite
of low for example 12 $/kg biomass cost being fulfilled.
To contribute remarkably to worlds energy demand, the
amounts of algae produced have to be increased by another order
of magnitude. Estimated areal productivities are in the range of
approximately 85 kg biodiesel per hectare and day [82]. Here
the idea of the biorefinery becomes difficult due to the given
volume structure of the markets. Possible side products such as
carotenoids in addition to the fuel would come in so tremendous
amounts that no market is big enough to sell the products.
Assuming a normal macromolecular composition of an algae
cell with for example 30% protein would by far overfeed the
world, a strange idea. The first way out of this dilemma is to also
use proteins, nucleic acids, and carbohydrates energetically, par-
tially to cover the energy demand of the production site itself and
to recycle nitrogen and phosphorus. The second way is to change
the stoichiometry of the growth pattern of the cell. As nothing
is for free, it has to be determined, on what energetic costs that
is possible. Oil production to high contents is for example done
by the cell with a lower energetic efficiency than the formation
of carbohydrates, it takes longer and it is accompanied by cell
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stress [95]. It is a matter of optimization, which oil content is
finally most profitable, it is not necessarily the highest possi-
ble [51, 96]. In the last consequence, only a complete change of
the stoichiometry toward biomass stagnation at growth rate zero
and channeling the carbon flux solely to the desired energetic
chemicals will result in a viable business model. Here again the
secretion of extracellular compounds and the new green chem-
istry are of highest interest. To achieve this goal on the biological
level, keeping the energetic efficiencies anticipated nowadays will
need new ideas and much further research.
4.3 Adaptation to cultural realities and creating
societal benefits
Scientific research is the gate between the biological poten-
tial of the algae and the supply of products in line with
the market. In the last few years visibility of microalgal
biotechnology to the general public was generated by press
releases such as Venture Beat: $ 144 m new investment in
algae (http://venturebeat.com/2012/04/02/sapphire-energy-
gets-144m-in-its-fuel-tank-to-turn-algae-into-gasoline)or
. . . a federation which will bring together all the players
in the development of micro-algae in France. A budget
of US$ 210 million for 10 years has been estimated . . .
(http://algaetec.com.au/2012/05/algae-news-wrap-up-a-
french-federation-for-micro-algae-development-secures-
funding/). Indeed, the amount of money that is spent now
for research on microalgae goes into roughly estimated 100
millions per year. While it may be more focused on large flagship
initiatives in the United States, it is not much less in Asia or
the European Union. Large scientific consortia have been estab-
lished to integrate different scientific fields of basic and applied
sciences. Pilot and demonstration plants are emerging all over
the world. A large European research consortium has summa-
rized its vision on debottlenecking microalgal metabolism to
go for a smarter and more efficient production process.
For the time being, money from microalgae is made mainly
as healthy food and feed supplements in comparably small mar-
kets [97]. A few years ago, normal consumers and even scientists
did not believe that algae products could be an ingredient of
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Figure 9. How will microalgal biotechnology change
life in rich and poor countries? (Picture kindly pro-
vided by Dr. Willy Brchle.)
the daily diet in really large volumes. Probably the imagination
was a greenish, strange smelling something on the plate that
the typical American or European consumer would not like.
This assessment came without considering that even now prod-
ucts from naturally growing macroalgae (polysaccharides such
as Alginate or Agar-Agar, E401E406) are processed in standard
foods. In the meantime, big food companies have already devel-
oped and patented processes to gain color, smell, and tasteless
protein extracts from microalgae. Even if the application of these
technologies in large volumes will take some time to be applied
in developed countries, microalgae will contribute remarkably
to supplying healthy food to a growing and hungry world pop-
ulation [98].
The current hype in microalgal biotechnology is driven by
the need for sustainable energy. The main driver is the aircraft
industry. This is due to the fact that airplanes cannot fly with
hydrogen or electricity from batteries but need liquid fuels of
high energy density. Show flights with different airplanes and
algae fuels have impressed the public but are only the tip of the
iceberg of current industrial activities. How long will it take until
algal biofuel is commercially available? Several large microalgal
companies claim they will achieve technology readiness in the
near term. However, algal mass production will not emerge in a
day. Research and application have to move forward over several
stepping stones characterized by larger areas but lower costs
[99, 100]. Today, we produce high-value products in only a few
thousand tons volume. Market prices for algae reach 50 $/kg
dry mass or more. The next steps will be food, feed for cattle
and aquaculture. Especially this last item is intensively addressed
facing empty oceans and the need for fish meal from natural
catch to get healthy fish in aquaculture. Bulk products are going
to follow where the final price of the product may not exceed
1 or 2 $/kg. For a remarkable contribution thousands of square
kilometers of algae production facilities are necessary. Only by
learning from these applications it will be possible to reach really
large amounts for biofuel production, where the process has to
be completely energy-neutral and the costs of the final product
such as biodiesel can compete with other sources.
However, integration of microalgal biotechnology into
our society must balance risks, opportunities, and ethical

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www.els-journal.com Eng. Life Sci. 2014, 14, 560573
considerations in the different fields of applications. Everybody
will welcome more food and fuel. But there are other questions
about working places and safety. Rich countries will profit from
energy availability and from selling cultivation technology. The
sun shines nearly everywhere, so there is an educated guess that
poor countries can also make business cases and create working
places (Fig. 9). Everybody will hopefully profit from less political
dependency on a few oil-exporting countries.
The authors have declared no conflict of interest.
5 References
[1] Borowitzka, M. A., High-value products from microalgae
their development and commercialisation. J. Appl. Phycol.
2013, 25, 743756.
[2] Borowitzka, M. A., Moheimani, N. R., Sustainable biofuels
from algae. Mitig. Adapt. Strat. Global Change 2013, 18, 13
25.
[3] Pulz, O., Gross, W., Valuable products from biotechnology of
microalgae. Appl. Microbiol. Biotechnol. 2004, 65, 635648.
[4] Harun, R., Singh, M., Forde, G. M., Danquah, M. K., Bio-
process engineering of microalgae to produce a variety of
consumer products. Renew. Sust. Energ. Rev. 2010, 14, 1037
1047.
[5] Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A.,
Commercial applications of microalgae. J. Biosci. Bioeng.
2006, 101, 8796
[6] Wijffels, R. H., Barbosa, M. J., An outlook on microalgal
biofuels. Science 2010, 329, 796799.
[7] Norton, T. A., Melkonian, M., Andersen, R. A., Algal biodi-
versity. Phycologia 1996, 35, 308326
[8] Metting, F. B., Biodiversity and application of microalgae. J.
Ind. Microbiol. Biotechnol. 1996, 17, 477489.
[9] Walker, T. L., Purton, S., Becker, D. K., Collet, C., Microalgae
as bioreactors. Plant Cell Rep. 2005, 24, 629641.
[10] Rodolfi, L., Zittelli, G. C., Bassi, N., Padovani, G. et al., Mi-
croalgae for oil: Strain selection, induction of lipid synthesis
and outdoor mass cultivation in a low-cost photobioreactor.
Biotechnol. Bioeng. 2009, 102, 100112.
[11] Mooij, P. R., Stouten, G. R., Tamis, J., van Loosdrecht, M. C.
M. et al., Survival of the fattest. Energy Environ. Sci. 2013, 6,
34043406.
[12] Gudin, C.,Chaumont, D., Cell fragility the key problem
of microalgae mass production in closed photobioreactors.
Bioresour. Technol. 1991, 38, 145151.
[13] Sobczuk, T., Camacho, F., Grima, E., Chisti, Y., Effects of agi-
tation on the microalgae Phaeodactylum tricornutum and Por-
phyridium cruentum. Bioprocess Biosyst. Eng. 2006, 28, 243
250.
[14] Renaud, S. M., Thinh, L. V., Lambrinidis, G., Parry, D.
L., Effect of temperature on growth, chemical composition
and fatty acid composition of tropical Australian microalgae
grown in batch cultures. Aquaculture 2002, 211, 195214.
[15] Jacobi, A., Bucharsky, E. C., Guenter, S. K., Habisreuther, P.
et al., The application of transparent glass sponge for improve-
ment of light distribution in photobioreactors. J. Bioprocess
Biotech. 2012, 2, 28.

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
[16] Beckmann, J., Lehr, F., Finazzi, G., Hankamer, B. et al., Im-
provement of light to biomass conversion by de-regulation of
light-harvesting protein translation in Chlamydomonas rein-
hardtii. J. Biotechnol. 2009, 142, 7077.
[17] Ma, J., Zheng, R., Xu, L., Wang, S., Differential sensitivity of
two green algae, Scenedesmus obliqnus and Chlorella pyrenoi-
dosa, to 12 pesticides. Ecotoxicol. Environ. Saf. 2002, 52, 5761
[18] Kropat, J., Hong-Hermesdorf, A., Casero, D., Ent, P. et al., A
revised mineral nutrient supplement increases biomass and
growth rate in Chlamydomonas reinhardtii. Plant J. 2011, 66,
770780.
[19] Rosch, C., Skarka, J., Wegerer, N., Materials flow modeling of
nutrient recycling in biodiesel production from microalgae.
Bioresour. Technol. 2012, 107, 191199.
[20] Klok, A. J., Verbaanderd, J. A., Lamers, P. P., Martens, D.
E. et al., A model for customising biomass composition in
continuous microalgae production. Bioresour. Technol. 2013,
146, 89100.
[21] Molina Grima, E., Belarbi, E. H., Acien Fernandez, F. G.,
Robles Medina, A. et al., Recovery of microalgal biomass and
metabolites: Process options and economics. Biotechnol. Adv.
2003, 20, 491515.
[22] Sukenik, A., Shelef, G., Algal autoflocculationverification
and proposed mechanism. Biotechnol. Bioeng. 1984, 26, 142
147.
[23] Park, J. B. K., Craggs, R. J., Shilton, A. N., Recycling algae to
improve species control and harvest efficiency from a high
rate algal pond. Water Res. 2011, 45, 66376649.
[24] Hashimoto, S., Furukawa, K., Nutrient removal from sec-
ondary effluent by filamentous algae. J. Ferment. Bioeng. 1989,
67, 6269.
[25] Esson, D., Wood, S., Packer, M., Harnessing the self-
harvesting capability of benthic cyanobacteria for use in ben-
thic photobioreactors. AMB Express 2011, 1, 19.
[26] Bruno, L., Di Pippo, F., Antonaroli, S., Gismondi, A.
et al., Characterization of biofilm-forming cyanobacteria for
biomass and lipid production. J. Appl. Microbiol. 2012, 113,
10521064.
[27] Johnson, M. B., Wen, Z., Development of an attached microal-
gal growth system for biofuel production. Appl. Microbiol.
Biotechnol. 2009, 85, 525534
[28] Liu, T. Z., Wang, J. F., Hu, Q., Cheng, P. F. et al., Attached cul-
tivation technology of microalgae for efficient biomass feed-
stock production. Bioresour. Technol. 2013, 127, 216222.
[29] Pruvost, J., Photobioreactor Engineering for Solar Microal-
gae Cultivation: Methodology and Applications, AlgnChem,
Montpellier, France 2014.
[30] Blanken, W., Wijffels, R. H., Biofilm Growth of Chlorella
Sorokiniana in the Algadisk System: A Rotating Biological Con-
tactor Based Photobioreactor, Young Algaeneers Symposium,
Narbonne, France 2014.
[31] Gunther, A., Jakob, T., Goss, R., Konig, S. et al., Methane
production from glycolate excreting algae as a new concept
in the production of biofuels. Bioresour. Technol. 2012, 121,
454457.
[32] Boelee, N. C., Temmink, H., Janssen, M., Buisman, C.
J. N. et al., Scenario analysis of nutrient removal from
municipal wastewater by microalgal biofilms. Water 2012, 4,
460473.
571
www.els-journal.com Eng. Life Sci. 2014, 14, 560573
[33] Mulbry, W., Kondrad, S., Pizarro, C., Kebede-Westhead, E.,
Treatment of dairy manure effluent using freshwater algae:
Algal productivity and recovery of manure nutrients using
pilot-scale algal turf scrubbers. Bioresour. Technol. 2008, 99,
81378142.
[34] Christenson, L., Sims, R., Production and harvesting of mi-
croalgae for wastewater treatment, biofuels, and bioproducts.
Biotechnol. Adv. 2011, 29, 686702.
[35] Bonente, G., Formighieri, C., Mantelli, M., Catalanotti, C.
et al., Mutagenesis and phenotypic selection as a strategy to-
ward domestication of Chlamydomonas reinhardtii strains for
improved performance in photobioreactors. Photosynthesis
Res. 2011, 108, 107120.
[36] Borowitzka, M. A., Commercial production of microalgae:
Ponds, tanks, tubes and fermenters. J. Biotechnol. 1999, 70,
313321.
[37] Molina Grima, E., Fernandez, F. G. A., Garca Camacho, F.,
Chisti, Y., Photobioreactors: Light regime, mass transfer, and
scaleup. J. Biotechnol. 1999, 70, 231247.
[38] Lee, Y. K., Microalgal mass culture systems and methods:
Their limitation and potential. J. Appl. Phycol. 2001, 13, 307
315.
[39] Posten, C., Design principles of photo-bioreactors for
cultivation of microalgae. Eng. Life Sci. 2009, 9,
165177.
[40] Bruggeman, A. J., Kuehler, D., Weeks, D. P., Evaluation of three
herbicide resistance genes for use in genetic transformations
and for potential crop protection in algae production. Plant
Biotechnol. J. 2014, doi: 10.1111/pbi.12192.
[41] Soletto, D., Binaghi, L., Ferrari, L., Lodi, A. et al., Effects
of carbon dioxide feeding rate and light intensity on the fed-
batch pulse-feeding cultivation of Spirulina platensis in helical
photobioreactor. Biochem. Eng. J. 2008, 39, 369375.
[42] Burger, D. E., Jett, J. H., Mullaney, P. F., Extraction of morpho-
logical features from biological models and cells by Fourier
analysis of static light scatter measurements. Cytometry 1982,
2, 327336.
[43] Schreiber, U., Pulse-amplitude-modulation (PAM) fluorom-
etry and saturation pulse method: An overview, in: Papa-
georgiou, G. C., Govindjee (Eds.), Chlorophyll a Fluorescence,
Springer, The Netherland s 2004, pp. 279319.
[44] Berenguel, M., Rodriguez, F., Acien, F. G., Garcia, J. L., Model
predictive control of pH in tubular photobioreactors. J. Process
Control 2004, 14, 377387.
[45] Ugwu, C. U., Aoyagi, H., Uchiyama, H., Photobioreactors for
mass cultivation of algae. Bioresour. Technol. 2008, 99, 4021
4028.
[46] Lee, A. K., Lewis, D. M., Ashman, P. J., Microbial flocculation,
a potentially low-cost harvesting technique for marine mi-
croalgae for the production of biodiesel. J. Appl. Phycol. 2009,
21, 559567.
[47] Evans, L. B., Bioprocess simulation: A new tool for process
development. Nat. Biotechnol. 1988, 6, 200203.
[48] Petrides, D., Cooney, C. L., Evans, L. B., Field, R. P. et al.,
Bioprocess simulation: An integrated approach to process de-
velopment. Comput. Chem. Eng. 1989, 13, 553561.
[49] Zhou, Y. H., Titchener-Hooker, N. J., Visualizing integrated
bioprocess designs through windows of operation. Biotech-
nol. Bioeng. 1999, 65, 550557.
572
www.biotecvisions.com
[50] Woodley, J. M., Titchener-Hooker, N. J., The use of windows
of operation as a bioprocess design tool. Bioprocess Biosyst.
Eng. 1996, 14, 263268.
[51] Pruvost, J., van Vooren, G., Le Gouic, B., Couzinet-Mossion,
A. et al., Systematic investigation of biomass and lipid pro-
ductivity by microalgae in photobioreactors for biodiesel ap-
plication. Bioresour. Technol. 2011, 102, 150158.
[52] Doucha, J., Lvansky, K., Productivity, CO2/O2 exchange and
hydraulics in outdoor open high density microalgal (Chlorella
sp.) photobioreactors operated in a Middle and Southern Eu-
ropean climate. J. Appl. Phycol. 2006, 18, 811826.
[53] Cuhel, R. L., Ortner, P. B., Lean, D. R. S., Night synthesis of
protein by algae. Limnol. Oceanogr. 1984, 29, 731744.
[54] Nagle, N., Lemke, P., Production of methyl ester fuel from
microalgae. Appl. Biochem. Biotechnol. 1990, 2425, 355361.
[55] Miao, X., Wu, Q., Biodiesel production from heterotrophic
microalgal oil. Bioresour. Technol. 2006, 97, 841846.
[56] Ehimen, E. A., Sun, Z. F., Carrington, C. G., Variables affecting
the in situ transesterification of microalgae lipids. Fuel 2010,
89, 677684.
[57] Geresh, S., Mamontov, A., Weinstein, J., Sulfation of extracel-
lular polysaccharides of red microalgae: Preparation, charac-
terization and properties. J. Biochem. Biophys. Methods 2002,
50, 179187.
[58] Patel, A. K., Laroche, C., Marcati, A., Ursu, A. V. et al.,
Separation and fractionation of exopolysaccharides from
Porphyridium cruentum. Bioresour. Technol. 2013, 145, 345
350.
[59] Frenz, J., Largeau, C., Casadevall, E., Hydrocarbon recovery
by extraction with a biocompatible solvent from free and
immobilized cultures of Botryococcus braunii. Enzyme Microb.
Technol. 1989, 11, 717724.
[60] Samor, C., Torri, C., Samor, G., Fabbri, D. et al., Extraction
of hydrocarbons from microalga Botryococcus braunii with
switchable solvents. Bioresour. Technol. 2010, 101, 32743279.
[61] Dornenburg, H., Knorr, D., Semicontinuous processes for
anthraquinone production with immobilized Cruciata glabra
cell cultures in a three-phase system. J. Biotechnol. 1996, 50,
5562.
[62] Lehr, F., Morweiser, M., Rosello Sastre, R., Kruse, O. et al.,
Process development for hydrogen production with Chlamy-
domonas reinhardtii based on growth and product formation
kinetics. J. Biotechnol. 2012, 162, 8996.
[63] Eroglu, E., Melis, A., Photobiological hydrogen production:
Recent advances and state of the art. Bioresour. Technol. 2011,
102, 84038413.
[64] Kleinegris, D. M. M., Janssen, M., Brandenburg, W. A., Wijf-
fels, R. H., Two-phase systems: Potential for in situ extraction
of microalgal products. Biotechnol. Adv. 2011, 29, 502507.
[65] Hejazi, M. A., Kleinegris, D., Wijffels, R. H., Mechanism of
extraction of -carotene from microalga Dunaliellea salina in
two-phase bioreactors. Biotechnol. Bioeng. 2004, 88, 593600.
[66] Kleinegris, D. M. M., Janssen, M., Brandenburg, W. A., Wi-
jffels, R. H., Continuous production of carotenoids from
Dunaliella salina. Enzyme Microb. Technol. 2011, 48, 253259.
[67] Marchal, L., Mojaat-Guemir, M., Foucault, A., Pruvost,
J., Centrifugal partition extraction of -carotene from
Dunaliella salina for efficient and biocompatible recovery of
metabolites. Bioresour. Technol. 2013, 134, 396400.

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.els-journal.com Eng. Life Sci. 2014, 14, 560573
[68] Glembin, P., Kerner, M., Smirnova, I., Cloud point extraction
of microalgae cultures. Separ. Purif. Method 2013, 103, 2127.
[69] Decker, E. L., Reski, R., The moss bioreactor. Curr. Opin. Plant
Biol. 2004, 7, 166170.
[70] Lucumi, A., Posten, C., Establishment of long-term perfusion
cultures of recombinant moss in a pilot tubular photobiore-
actor. Process Biochem. 2006, 41, 21802187.
[71] Cerff, M., Morweiser, M., Dillschneider, R., Michel, A. et al.,
Harvesting fresh water and marine algae by magnetic sepa-
ration: Screening of separation parameters and high gradient
magnetic filtration. Bioresour. Technol. 2012, 118, 289295.
[72] Schugerl, K., Hubbuch, J., Integrated bioprocesses. Curr.
Opin. Microbiol. 2005, 8, 294300.
[73] Hawkins, R. L., Nakamura, M., Expression of human growth
hormone by the eukaryotic alga Chlorella. Curr. Microbiol.
1999, 38, 335341.
[74] Lauersen, K. J., Berger, H., Mussgnug, J. H., Kruse, O., Efficient
recombinant protein production and secretion from nuclear
transgenes in Chlamydomonas reinhardtii. J. Biotechnol. 2013,
167, 101110.
[75] Hempel, F., Lau, J., Klingl, A., Maier, U. G., Algae as protein
factories: Expression of a human antibody and the respective
antigen in the diatom Phaeodactylum tricornutum. PLoS One
2011, 6, 17.
[76] Lauersen, K. J., Vanderveer, T. L., Berger, H., Kaluza, I.
et al., Ice recrystallization inhibition mediated by a nuclear-
expressed and -secreted recombinant ice-binding protein in
the microalga Chlamydomonas reinhardtii. Appl. Microbiol.
Biotechnol. 2013, 110.
[77] Robertson, D. E., Jacobson, S. A., Morgan, F., Berry, D. et al.,
A new dawn for industrial photosynthesis. Photosynth. Res.
2011, 107, 269277.
[78] Harun, R., Danquah, M. K., Forde, G. M., Microalgal biomass
as a fermentation feedstock for bioethanol production. J.
Chem. Technol. Biot. 2010, 85, 199203.
[79] Croft, M. T., Lawrence, A. D., Raux-Deery, E., Warren, M. J.
et al., Algae acquire vitamin B12 through a symbiotic rela-
tionship with bacteria. Nature 2005, 438, 9093.
[80] Mouget, J. L., Dakhama, A., Lavoie, M. C., Noue, J., Algal
growth enhancement by bacteria: Is consumption of photo-
synthetic oxygen involved? FEMS Microbiol. Ecol. 2006, 18,
3543.
[81] Grossart, H., Simon, M., Interactions of planktonic algae and
bacteria: effects on algal growth and organic matter dynamics.
Aquat. Microb. Ecol. 2007, 47, 163176.
[82] Wilhelm, C., The biological perspective ideas from new green
chemistry concepts for improving the performance of mi-
croalgae. Technikfolgenabschatzung Theorie und Praxis 2012,
21, 4653.
[83] Rodrguez-Zavala, J. S., Ortiz-Cruz, M. A., Mendoza-
Hernandez, G., Moreno-Sanchez, R., Increased synthesis of
-tocopherol, paramylon and tyrosine by Euglena gracilis un-
der conditions of high biomass production. J. Appl. Microbiol.
2010, 109, 21602172.
[84] Quesada, L. A., Delustig, E. S., Marechal, L. R., Belocopitow,
E., Antitumor activity of paramylon on sarcoma-180 in mice.
Gann 1976, 67, 455459.
[85] Yang, J., Xu, M., Zhang, X., Hu, Q. et al., Life-cycle anal-
ysis on biodiesel production from microalgae: Water foot-

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
print and nutrients balance. Bioresour. Technol. 2011, 102,
159165.
[86] Boelee, N. C., Temmink, H., Janssen, M., Buisman, C. J.
N. et al., Nitrogen and phosphorus removal from munici-
pal wastewater effluent using microalgal biofilms. Water Res.
2011, 45, 59255933.
[87] Olaizola, M., Microalgal removal of CO2 from flue
gases: Changes in medium pH and flue gas composi-
tion do not appear to affect the photochemical yield
of microalgal cultures. Biotechnol. Bioproc. Eng. 2003, 8,
360367.
[88] Chiu, S.-Y., Kao, C.-Y., Huang, T.-T., Lin, C.-J. et al., Mi-
croalgal biomass production and on-site bioremediation of
carbon dioxide, nitrogen oxide and sulfur dioxide from flue
gas using Chlorella sp. cultures. Bioresour. Technol. 2011, 102,
91359142.
[89] Tredici, M. R., Sampiertro, G., Rodolfi, L., Biondi, N., Mi-
croalgae for biofuels and food: Integration of photovoltaic
cells in a green wall panel (GWP) photobioreactor improves
the EROI of algal biomass production. Algal Biomass, Biofuels
and Bioproducts Toronto, Canada, 2013.
[90] Nobre, B. P., Villalobos, F., Barragan, B. E., Oliveira, A. C.
et al., A biorefinery from Nannochloropsis sp. microalga
Extraction of oils and pigments. Production of biohydrogen
from the leftover biomass. Bioresour. Technol. 2013, 135, 128
136.
[91] Yen, H.-W., Hu, I.C., Chen, C.-Y., Ho, S.-H. et al., Microalgae-
based biorefinery from biofuels to natural products. Biore-
sour. Technol. 2013, 135, 166174.
[92] Kamm, B., Kamm, M., Principles of biorefineries. Appl. Mi-
crobiol. Biotechnol. 2004, 64, 137145.
[93] Subhadra, B., Grinson, G., Algal biorefinery-based indus-
try: An approach to address fuel and food insecurity
for a carbon-smart world. J. Sci. Food Agric. 2011, 91,
213.
[94] OPEC, World Oil Outlook 2013, OPEC Secretariat, Vienna,
Austria 2013.
[95] Blatti, J. L., Michaud, J., Burkart, M. D., Engineering fatty
acid biosynthesis in microalgae for sustainable biodiesel. Curr.
Opin. Chem. Biol. 2013, 17, 496505.
[96] Dillschneider, R., Steinweg, C., Rosello-Sastre, R., Posten,
C., Biofuels from microalgae: Photoconversion efficiency
during lipid accumulation. Bioresour. Technol. 2013, 142,
647654.
[97] Griehl, C., Bieler, S., Algen: Rohstoffe fur Gesundheit,
Schonheit und Energie. Nachrichten aus der Chemie 2011,
59, 942947.
[98] Draaisma, R. B., Wijffels, R. H., Slegers, P. M., Brentner, L.
B. et al., Food commodities from microalgae. Curr. Opin.
Biotechnol. 2013, 24, 169177.
[99] Sevigne Itoiz, E., Fuentes-Grunewald, C., Gasol, C. M.,
Garces, E. et al., Energy balance and environmental im-
pact analysis of marine microalgal biomass production for
biodiesel generation in a photobioreactor pilot plant. Biomass.
Bioenerg. 2012, 39, 324335.
[100] Slegers, P. M., van Beveren, P. J. M., Wijffels, R. H., van
Straten, G. et al., Scenario analysis of large scale algae pro-
duction in tubular photobioreactors. Appl. Energ. 2013, 105,
395406.
573