C H A P T E R

16
New strategies enhancing feasibility of
microalgal cultivations
Fabrizio Di Caprio, Pietro Altimari, Francesca Pagnanelli*
Department of Chemistry, University Sapienza of Rome, Rome, Italy
* Corresponding author. e-mail address: Francesca.Pagnanelli@uniroma1.it

1. Introduction develop an industry able to use these resources

as feedstocks to obtain several organic molecules
Microalgae are unicellular microorganisms has strongly attracted the interest of scientific re-
that live in nature by exploiting photosynthetic searchers and industrialists [4]. Although these
metabolism. Microalgae is the name by which general features are owned also by terrestrial
eukaryotic photosynthetic microorganisms are plants, microalgae have some properties that
generally indicated, while prokaryotic photosyn- make them more promising as future biomass
thetic microorganisms are named cyanobacteria sources with respect to terrestrial plants:
(green-blue algae). For simplicity, in this chapter
- Microalgae have higher photosynthetic
the term “microalgae” will refer to both. It is esti-
efficiency (maximum value w 10%) with
mated that up to 200,000 species of microalgae
respect to terrestrial plants (maximum
may exist [1]. They live in nature in a large vari-
value w 5%) [5e8].
ety of different environments such as seas, lakes,
- Microalgae can reach higher biomass
rivers, glaciers, wastewaters, etc.
productivity (50e70 t/ha per year) with
Exploiting their metabolism, microalgae can
respect to terrestrial plants (10e20 t/ha per
use inorganic carbon (CO2, HCOe 2

3 , and CO3 ),
year) [1,9,10].
light, water, and simple mineral salts (PO3
4 ,
- Microalgae can grow on nonarable lands and
NO
3 , Ca 2þ
, Mg 2þ
, etc.) to synthetize complex
in the sea [11].
organic molecules such as proteins, fatty acids,
- Microalgae can grow in closed systems and
polysaccharides, carotenoids, and others [2]
by using wastewaters and saltwater, thus
(Fig. 16.1). These molecules can be used for a
reducing considerably freshwater
large variety of applications such as biofuels,
consumption [9].
bioplastics, feed, and food [3].
CO2 and light are renewable resources widely However, despite these potentialities, the
available; consequently, the possibility to biomass produced by terrestrial plant is still the

Catalysis, Green Chemistry and Sustainable Energy

https://doi.org/10.1016/B978-0-444-64337-7.00016-1 287 Copyright © 2019 Elsevier B.V. All rights reserved.
288 16. New strategies enhancing feasibility of microalgal cultivations

FIGURE 16.1 Schematic illustration of microalgae photosynthetic metabolism.

main biomass available as feedstock for the in-
dustry. The worldwide microalgae biomass pro-
duction was estimated 5000 t/y for Spirulina in
2013 and 2000 t/y for Chlorella in 2003 [12].
This amount is negligible with respect to
biomass coming from agriculture. It corresponds
to about 400e500 ha of maize cultivated land,
which is comparable with the average produc-
tion of just two average-sized farms in the
United States [13]. This limited production is
mainly given by the high cost associated with
microalgae biomass production. At the state of
the art, with the best available technologies, the
minimum cost achievable for microalgae
biomass production is between 3 and 10 V per
kg of dry microalgae biomass, according to esti-
mates published in 2016 [11,14,15]. Costs lower
than 3 V/kg have been also reported in different
works; however, these latter derive from as-
sumptions and forecasts that still need to be
experimentally verified [14e16]. The elevated
cost is given by the application of expensive
cultivation systems (mainly open ponds, flat
panel and tubular photobioreactors) for reactors
installation and for continuous power supply
(required for cultivation and harvesting) that
are not balanced by the biomass productivity.
The most widespread agricultural crops are
less productive with respect to microalgae, but
the cultivation systems used are enough cheap
to ensure lower production costs. For example,
the price associated with some of the main vege-
table commodities is about 0.5 V/kg for palm
oil, 0.2 V/kg for corn, and 0.3 V/kg for soybean
(these three can be considered the main vege-
table commodities for lipids, carbohydrates,
and proteins, respectively) (Index Mundi,
2018). Due to the high cost associated, currently
microalgae biomass production is mainly carried
out for high added value products (Table 16.1)
such as microalgal powder, tablets, supple-
ments, and ingredients for human and animal
nutrition.
For example, docosahexaenoic acid (DHA) is
an omega-3 fatty acid produced mainly by het-
erotrophic cultivation of Schizochytrium sp. in
different plants located in the United States and
France. Haematococcus pluvialis is cultivated by
Cyanotech (in Hawaii) and Algatech (Israel) to
produce an oleoresin enriched in astaxanthin,
which is obtained by CO2 supercritical extraction
from dried biomass. Chlorella and Spirulina species

IV. Selected examples and case history

 2. Development of biorefineries 289
TABLE 16.1 Main companies involved in industrial production of microalgae biomass and derivates.

Plant
Company name Species cultivated size Products Reactor type References

Earthrise Spirulina (Arthrospira 108 ha Powder, tablets and Open ponds http://earthrise.
platensis) phycocyanin extracts com/
Taiwan Chlorella Chlorella sorokiniana 2.6 ha Powder, tablets, and Open ponds http://www.
Manufacturing chlorella extracts taiwanchlorella.com
Company
otze Chlorella vulgaris
Roquette Kl€ 500 km Powder Tubular www.algomed.de/
GmbH & co. KG glass photobioreactors en
tubes
Algatechnologies Haematococcus pluvialis 600 km Astaxanthin and Tubular https://www.
Ltd. and Phaeodactylum glass fucoxanthin photobioreactors algatech.com
tricornutum tubes

Cyanotech Haematococcus pluvialis 36 ha Spirulina tablets and Open ponds https://www.

and Spirulina encapsulated oleoresin cyanotech.com/
(Arthrospira platensis) enriched in astaxanthin
Phycom Chlorella sorokiniana and e Pellets, powder, and flakes Closed fermenters http://www.
Chlorella vulgaris phycom.eu
Solazyme Different Chlorella sp. e Oils and several food Closed fermenters http://
strains ingredients solazymeindustrials.
com/
Alltech Schizochytrium sp. e DHA oil Closed fermenters https://www.
alltech.com/

Fermentalg Schizochytrium sp. e DHA oil or pigments Mixotrophy using https://www.

mainly heterotrophy fermentalg.com/fr/
(fermenters þ flash)

are often placed on the market as a powder rich in 2. Development of biorefineries

proteins. Such powders are dried and sold as
pressed pills, flakes, or tablets for nutraceutical ap-
plications [12]. The powders have also applications
as animal protein replacement for vegan food
production (e.g., Terranostra Food s.r.l., Italy).
In the following sections the new promising
strategies to enhance the feasibility of microalgae
biomass production at larger industrial scale are
described. The described strategies are develop-
ment of biorefineries (Section 2), innovative pho-
tobioreactors (Section 3), wastewater utilization
as a source of nutrients (Section 4), and strain
improvement (Section 5).
The aim of this section is to describe the devel-
opment of a biorefinery, which is one of the most
promising approaches to enhance the feasibility
of microalgae processes. The concept of a bio-
refinery is defined in Subsection 2.1, then the
different products obtainable from microalgae
biomass are described in Subsection 2.2. Subse-
quently, in Subsection 2.3, the latest innovations
in the development of a biorefinery from micro-
algae are described, illustrating the shifting of
the processes from single end products to multi-
ple end products.

IV. Selected examples and case history

290 16. New strategies enhancing feasibility of microalgal cultivations
FIGURE 16.2 Comparison between a conventional process and an example of biorefinery.
2.1 Definition of biorefinery
With the term “biorefinery” a facility is indi-
cated, in which microalgae biomass is used as
feedstock to produce a large variety of different
products. The term derives from the conven-
tional petroleum refineries in which crude oil is
used as feedstock to produce a large variety of
different petrochemical derivatives (fuels, oils,
heat, solvents, plastics, etc.). It is expected that
the development of biorefineries may enhance
economic and environmental sustainability of
microalgae biomass production, through the
valorization of different components of the pro-
duced biomass. Until now, industrial processes
that produce microalgae have been designed to
obtain only a single product from a single
biomass. The biorefinery aims to change this
concept by shifting the process design from one
biomass / one product to one biomass / mul-
tiple products (Fig. 16.2).
2.2 Potential products obtainable by a
microalgae biorefinery
Hypothetically, every component of the
microalgae biomass may be exploited for a
IV. Selected examples and case history
different product. The main carbohydrates
(starch and cellulose) could be used to produce
bioplastics, biofuels, or food [17e19], while
some other carbohydrates (e.g., b-glucans) could
have applications as nutraceuticals or cosmetics
[20,21]. Microalgae proteins generally have a
good composition in amino acids, so they are
considered a promising future source of proteins
for human nutrition (especially in replacing ani-
mal proteins) and for feed (for livestock and
aquaculture) [22,23]. Microalgae proteins can
also be exploited to produce bioplastics, for
example, by processing directly dried biomass
rich in proteins [24], or to synthetize polyure-
thanes starting from amino acids [25]. Some pep-
tides, with health benefit properties, can be
produced by means of specific digestion [26].
Proteins include enzymes, which could be
extracted and used as catalyzers for a large vari-
ety of different reactions with applications in in-
dustry, for example, laccase and esterase
[27e29]. Proteins from microalgae showed supe-
rior surface activity and gelation behavior when
compared with whey proteins, with applications
in food industry [30,31].
Lipids from microalgae can be divided in
neutral lipids, mainly triacylglycerols (TAGs),
 2. Development of biorefineries
and polar lipids, mainly phospholipids [32].
Some microalgae, for example, Botryococcus brau-
nii, can accumulate hydrocarbons too [33]. Fatty
acids from microalgae can be used to produce
biofuels (biodiesel above all) [34], bioplastics
[35], and other products, similarly to what is
already done in the industry starting from con-
ventional vegetable oils such as palm oil, soy
oil, and others. Glycerol deriving from hydroly-
sis of TAGs and phospholipids can be recycled
as nutrients to improve microalgae productivity
[36]. Differently to the widespread vegetable
oils, microalgae oils are rich in omega-3 fatty
acids (for example DHA), which make them a
promising substitute of fish oil for aquaculture
and to face the issue of omega-3/omega-6 imbal-
ance in the human diet [37,38].
Pigments such as carotenoids (astaxanthin,
b-Carotene), xanthophylls (lutein, zeaxanthin),
chlorophylls, and phycocyanin also received
attention for their high added value (between
300 and 3000 V/kg) [3]. Some of them are
already extracted for nutraceutical application
(Table 16.1). They can also be used as pigments
for food and feed; for example, astaxanthin is
responsible for the red color of salmon [39,40]
and lutein of the yellow-orange color of egg
yolk [41].
2.3 Shifting microalgae processes from
single products to multiple products
The shifting from conventional microalgae
processes to biorefinery facilities needs several
changes and improvements. The main changes
required are these:
- the shifting of conventional cultivation
processes in which a single biochemical
target component is maximized to the
optimization of processes tailored to achieve
a biomass composition balanced for the
different biochemical target components;
- the shifting of the downstream processes of
the biomass from separation/purification/
IV. Selected examples and case history
291
transformation of a single target product to
multiple target products.
The two changes are strongly related because
they depend on each other. Technical, economic,
and environmental sustainability are factors that
determine the number of target products obtain-
able from a microalgae biorefinery. Currently,
downstream treatments of microalgae biomass
are carried out by adapting technologies previ-
ously developed for other applications. For this
reason, the sustainability of the downstream
treatment is strongly limited, and a large part
of the companies sell biomass that is just dried
and pressed (Table 16.1).
To extract different compounds from the
same starting biomass, the extraction treat-
ments used for every compound should be
mild enough to avoid the degradation of the
other compounds. Much research has been con-
ducted to optimize the extraction of different
compounds from microalgae biomass; howev-
er, a large part of these works was carried out
by focusing on a single product. For example,
a study was carried out for the optimization of
lipid extraction and another one for the optimi-
zation of protein extraction, instead of carrying
out a single study to optimize both. The infor-
mation produced from these works is essential
for the development of a biorefinery, but it is
not enough. In fact, the preliminary information
achieved by the studies focusing on single prod-
ucts should be successively exploited to carry
out further studies in which extraction of
different compounds is optimized in a single
process. In this direction, difference strategies
have been proposed (Fig. 16.3).
All the downstream processes have to face
initially with the separation of the biomass
from water. Water is the solvent in which micro-
algae carry out their reactions, namely the sol-
vent in which they grow. In photoautotrophic
cultivations, water is generally between 99.9%
and 99.5% of the microalgae culture [42], while
in heterotrophic cultivations, it is generally
292 16. New strategies enhancing feasibility of microalgal cultivations

FIGURE 16.3 Illustration of the main strategies described for separation of different biomolecules from microalgae
biomass.

between 99.1% and 90% [43]. A large part of wa-
ter can be removed by the preliminary harvest-
ing phase, generally achievable by filtrations,
sedimentation, flocculation, or centrifugation
[44]. The parameters of the harvesting are mainly
determined by the biomass cultivation condition
(microalgae strain, final biomass concentration
achieved, etc.). Through harvesting, biomass
reaches a water content between 99% and 70%,
depending on the technology used, with an en-
ergy consumption between 0.01 and 8 kWh/kg
[15,44]. After harvesting, a large part of the pro-
posed processes includes a drying phase, by
which water content is reduced below 5%.
Freeze-drying and spray drying are the most
used technical methods to dry microalgae
biomass before compound extraction. However,
the drying process is also a very energy-intensive
treatment. Energy consumed for freeze-drying
has been estimated by some authors in the range
of 5e40 kWh/kg, corresponding to about 1e6
V/kg [45], while some other authors estimated
a lower cost for biomass drying corresponding
to about 0.18 V/kg [15]. Due to the impact of
the drying phase, there is a recent growing inter-
est toward the development of wet processes in
which extractions are carried out directly on
wet biomass [46].
The general strategy followed in the bio-
refinery is the cascade extraction approach, by
which different compounds are extracted by us-
ing sequential extractions and separation phases.
The intracellular molecules have to be extracted
and separated by means of a partial or total alter-
ation of cell structure. These alterations can be
more or less strong, in function of the used con-
ditions. In general, it can be considered that the
kinetic of the extraction increases by increasing
the cell destruction efficiency. In fact, when the
cells are still intact, or just partially broken, the
kinetic is strongly limited by the diffusion
gradient inside cells (or cell aggregates) and by
cellular layers (cell wall, cell membranes) that
obstruct molecule movement.

IV. Selected examples and case history

 2. Development of biorefineries
The gentlest alterations are those obtained by
some selective extractions that can be carried out
directly on live cells. In fact, it is possible to use
conditions mild enough to maintain the cells
alive. This kind of extraction is named milking
because of its similarity to the milking of cows.
Milking has been used to extract b-carotene
from (Dunaliella) salina [47], hydrocarbons from
B. braunii [48], and fatty acids from Nannochlorop-
sis sp. [49]. An essential condition for milking is
the adoption of biocompatible solvents. Zhang
and coworkers found that a partition coefficient
>5.5 (log Poct/wat) was required to be biocompat-
ible (nontoxic) for Nannochloropsis sp. cells [49].
However, it should be considered that milking
has kinetics and efficiency generally lower than
conventional extractions, and it is applicable
only for some molecules.
In the large parts of the processes investi-
gated, the approach followed was based on
sequential selective extractions coupled with
treatment for partial or total cell destruction
(death of cells). In every extraction condition,
some compounds are pulled out with a certain
selectivity from the residual biomass, by produc-
ing a solution enriched in such compounds.
To increase the extraction efficiency, different
technical methods have been studied and opti-
mized to achieve cell destruction. They can be
classified as mechanical methods such as high-
pressure homogenization, bead milling, ultraso-
nication, microwave, and pulsed electric fields
and nonmechanical methods such as
chemical and enzymatic treatments [45,50].
Chemical and enzymatic treatments are also
accompanied by reactions that can induce modi-
fication in the chemical properties of the
extracted molecules (e.g., hydrolysis). Some
extraction solvents, a large part of organic sol-
vents, are able themselves to break down cell
membranes by solubilizing membrane phospho-
lipids [51].
Data reported in the literature indicate that
the pretreatments carried out for cell disruption
require from 0.06 kWh/kg to 150 kWh/kg [50].
IV. Selected examples and case history
293
The large variability reported depends on the
technology used, microalgae species, biomass
concentration, suspension flow, pH, tempera-
ture, and also by the analytical method used to
assess cell destruction. Comparing the same
operative conditions, beat beater has been used
to achieve cell destruction of Chlorella vulgaris,
Neochloris oleoabundans, and Tetraselmis suecica
with a comparable energy consumption of
0.5 kWh/kg for all three strains [52]. Cell
destruction efficiency is generally quantified by
measuring cell disappearing by optical counting,
flowcytometry, or cell counter [52,53]. However,
that means that also with a reported 100% cell
destruction, there will be still a certain amount
of cell fragments (of variable size) in which
different molecules stay linked together, limiting
their extraction and separation. Consequently, it
is not easy to understand from literature data
what is exactly the amount of cell destruction
in the extracts, and data obtained can be strongly
influenced by the solid/liquid separation
method used after treatment.
Once the molecules are moved in solution,
they can be separated by treatments exploiting
conventional chemical and biochemical methods
(precipitation, solvent partition, chromatog-
raphy, etc.). Different studies have investigated
the possibility to separate fatty acids and lutein
starting from the extract obtained by lipid extrac-
tion with organic solvents. These works were
based on the different partition coefficient of
lutein and TAGs in an ethanol/hexane two-
phase system [54,55]. Biomass was first treated
with a cell destruction treatment coupled with
solvent extraction of lipids (e.g., by using ethanol
or directly hexane/ethanol mixture). This phase
could be coupled with alkaline treatment
because high OH
concentrations hydrolyze
lutein esters to free lutein, and at the same
time, OH
can enhance cell breaking (however
OH
should be carefully dosed for exposition
time and concentration to avoid lutein degrada-
tion) [41]. After lipid extraction, a two-phase
ethanol-hexane system can be obtained, in which
294 16. New strategies enhancing feasibility of microalgal cultivations
the lutein goes mainly in the ethanol phase,
while the TAGs go mainly in the hexane phase.
Pure lutein can be finally obtained from the
ethanol phase by means of precipitation induced
by water addition [56,57]. TAGs in the hexane
extract can be used to synthetize biodiesel by
converting fatty acids in their methyl esters by
transesterification.
The residual defatted biomass rich in proteins
and carbohydrates can be further valorized.
However, protein and carbohydrate separation
from defatted biomass is poorly investigated in
the literature.
The main alternatives investigated in the liter-
ature for the valorization of defatted biomass
concern the production of other biofuels: by hy-
drolysis (chemical or enzymatic) to produce sub-
strates for microbial fermentations for bioethanol
or biogas production [58,59] and by thermal and
hydrothermal treatments for biochar, gas, and
liquid fuels production [60,61]. Other investi-
gated applications include biosorption, by which
the defatted biomass was used as biosorbent for
heavy metal removal in contaminated water
[62].
Extractions carried out by using bead milling
in aqueous suspension allowed selective protein
extraction, with selectivity between 0.9 and 6.7
for proteins with respect to carbohydrates [52].
But it should be taken into account that this
study was conducted on nondefatted biomass.
Instead, in the processes investigated by
Ref. [63] and by Ref. [64], the microalgae biomass
was first treated with organic solvents to extract
lipids, and then proteins were extracted from
defatted biomass by using alkaline solutions.
However, although alkaline proteins extraction
was successfully used, for example, to remove
proteins from biomasses as rice flour [65,66],
when applied to microalgae biomass, it was
not very effective (only w 10% protein extrac-
tion) [63,64]. Desai and coworkers proposed a
process by which astaxanthin and proteins
were simultaneously extracted and separated
IV. Selected examples and case history
from Haematococcus pluvialis cells by exploiting
a microgel-stabilized ionic liquidewater emul-
sion. The astaxanthin was extracted in its ionic
liquid droplets, with a yield of 62%, while pro-
teins (90%) moved in the aqueous phase [67].
The protein extracts obtained by any extrac-
tion process can be processed by conventional
downstream treatments such as membrane
filtration and precipitation to produce protein
concentrates and protein hydrolysate [68,69].
3. Innovative photobioreactors
Many research works have been published
with the aim to investigate the feasibility of
microalgae cultivation, mainly for biofuel pro-
duction. In these works, different photoautotro-
phic configurations have been tested for
microalgae cultivation: however, not one has
reached the hoped performances, and the costs
to sustain microalgae biomass production are
yet too high for a large part of the expected ap-
plications (see Introduction section). The main
configurations tested in pilot-scale photoauto-
trophic plants were open ponds, tubular photo-
bioreactors (vertical and horizontal), and flat
panels. For all these tested configurations, the
biomass production cost was higher than 3
V/kg. The main factors affecting the cost of pro-
duced biomass were characteristics directly
linked to reactor design, such as operative costs
given by energy consumption and labor and
costs for depreciation [11,14e16,42]. Conse-
quently, new reactor configurations can poten-
tially improve the feasibility of microalgae
biomass production.
The aim of this section is to describe such new
reactor configurations. In Subsections 3.1 and
3.2, thin-layer photobioreactors and biofilm reac-
tors are described, respectively. In Subsection 3.3
other new promising configurations are
described, which have received less attention.
 3. Innovative photobioreactors
3.1 Thin-layer photobioreactors
One of the main bottlenecks limiting microal-
gae productivity in photobioreactors is light lim-
itation. To have high biomass productivity
inside reactors, microalgae should grow with a
fast growth rate (m w mmax) when they are at
high biomass concentration (dX/dt ¼ mX). How-
ever, when microalgae grow, increasing their
concentration (X), they progressively limit light
penetration because of the shading effect. As a
consequence, light available for microalgae cells
is progressively reduced inside reactors during
microalgae growth, and as cell concentration in-
creases the amount of photons supplied per cell
becomes progressively lower, making m < mmax
[70,71]. The higher the optical path of the reac-
tors is, then the lower the biomass concentration
at which light becomes limiting for microalgae
growth. The surface to volume ratio (S/V) of
photobioreactors decreases by decreasing the
optical path. The reactors widely tested for
microalgae cultivation at pilot scale have optical
paths between 0.02 and 0.3 m, corresponding to
S/V ratio between 5 and 100 m2/m3. By using
these configurations, microalgae biomass con-
centrations and biomass productivities typically
reached were between 0.2 and 5 g/L and be-
tween 0.01 and 1 g/L per day, respectively
[14,42,72].
Thin-layer photobioreactors can be defined as
reactors having an optical path 0.01 m. These
reactors have the potential to reach biomass con-
centrations and productivities much higher than
the widely tested configurations. That is because
light decrease inside a reactor follows an expo-
nential trend, so a difference of few millimeters
can lead to a relevant gain. However, the realiza-
tion of reactors with a very low optical path re-
quires one to face new issues that were not
encountered with the conventional configura-
tions. For instance, there is no space for the con-
ventional probes used for pH and temperature
control.
IV. Selected examples and case history
295
The thin-layer reactor designed by BCS Engi-
neering (Brno, Czech Republic) is a modified
open pond in which microalgae suspension is
moved by a centrifugal pump instead of a pad-
dle wheel. The suspension is circulated on the
surface of a glass sheet (sustained by a steel
frame) that is inclined 1.6%e1.7%, 18 m long,
and 1 m width. The suspension layer is main-
tained at an optical path between 6 and 8 mm,
with a 0.5 m/s speed. At the end of the sheet,
the culture is collected on a tank and recirculated
by the pump. During the day, CO2 is supplied to
the culture, whereas during the night the whole
suspension is maintained in the tank and
aerated. Water evaporation is balanced by
feeding water to the tank to keep a constant level
[73,74]. By using this configuration, microalgae
biomass concentrations between 8.9 and
29.7 g/L were achieved in about 15e30 days
[73]. A very similar configuration was tested by
Apel and coworkers too (5e5.6 mm optical
path), by achieving biomass concentrations up
to 50 g/L and biomass productivities of
2.4e2.9 g/L per day [75]. AlgoFilm is a photo-
bioreactor with an optical path of 1.5 to 2 mm.
It is similar to the reactors tested by
Refs. [73,75], but it is, in contrast, a closed sys-
tem, similar to an inclined flat panel, in which
the suspension is continuously recirculated
from the bottom part to the top part by using a
peristaltic pump. Chlorella vulgaris was culti-
vated in this configuration by reaching a
biomass concentration up to 30 g/L and a
biomass productivity up to 7 g/L per day.
Gifuni and coworkers investigated the perfor-
mances of an ultrathin flat photobioreactor
(UFP) with 3-mm optical path. The reactor was
built up by using three polymethylmethacrylate
panels (86  18 cm  0.5 mm) spaced out by two
silicone sheets having 3 mm thickness. The
panels were assembled in a sandwich structure
by obtaining a reactor volume of 0.3 L and a
back chamber used for cooling (by water recircu-
lation). Chlorella sorokiniana was cultivated in this
296 16. New strategies enhancing feasibility of microalgal cultivations
latter reactor until reaching 24 g/L of biomass
concentration and 11 g/L per day of biomass
productivity [76].
The studies carried out prove that by using
thin-layer photobioreactors microalgae concen-
tration and productivities comparable to those
achievable by heterotrophic cultivations can be
attained [43]. This property may reduce the
cost of the produced biomass with respect to
the conventional technologies, but cost analyses
are required from pilot-scale plants to consider
also the higher operative and installation costs
that will be likely given by the higher S/V ratio.
In this direction, preliminary data obtained by
Hulatt and Thoma indicate the net energy ratio
calculated for biomass produced by flat panel re-
actors becomes more favorable by optical path
reduction [77].
3.2 Biofilm photobioreactors
Biofilm bioreactors can be seen as a way to
work with an extremely reduced optical path.
In these reactors, microalgae cells live attached
to a surface within an amount of medium solu-
tion that is reduced to a minimum. The main ad-
vantages of the biofilm reactors are the high
photosynthesis efficiency and microalgae pro-
ductivity, which are obtained thanks to the facil-
itated gas exchange and light penetration.
Moreover, the costs for harvesting are almost
avoided because microalgae grow already sepa-
rated from the liquid, with water content
reduced to w70% [78]. Materials tested as sur-
faces for microalgae growth were cotton ropes,
filter paper or synthetic polymers, stainless-
steel woven meshes, and sanded polycarbonate
[79e81].
Biofilm reactors can be divided mainly in two
categories: submerged cultures and porous sub-
strate bioreactors (PSBRs).
The typical configuration for a submerged
culture biofilm reactor is made of a wheel
covered with a material on which microalgae
can easily grow attached and on which solution
IV. Selected examples and case history
medium can remain impregnated. The well ro-
tates remaining partially (about 40%e50%) sub-
merged in a tank containing medium solution
[79,80]. With this configuration, microalgae
grow attached to the external surface of the
wheel. About a half of the microalgae remain
submerged during cultivation, while another
half remain constantly covered by a thin water
film. Consequently, the configuration if these re-
actors still requires air/CO2 bubbling inside the
cultivation medium (in the tank), and they
require a further energy supply for wheel rota-
tion, and about a half of the microalgae are actu-
ally under limited condition for light penetration
and gas exchange [81].
To overcome these latter issues, PSBRs have
been studied recently. These particular biofilm
reactors are made of a porous material layer
(e.g., filter paper) on which microalgae grow
[82]. This layer separates microalgae cells from
the cultivation medium. In detail, cells grow on
one side of the layer of the porous material and
are directly exposed to air, while the cultivation
medium is only in contact with the other side of
the layer (Fig. 16.4). Water moves from one side
of the layer to the other side (the one with cells)
by passing through the porous material. The
movement of water can be given by different
mechanisms such as capillarity, difference of
pression, and water evaporation on the surface
exposed to air. Mineral salts move in the water
by the gradient diffusion between medium and
cells. PSBR configuration allows one to maintain
the advantages of the reduced optical path, it en-
hances gas exchange by cell exposition to air,
and at the same time, it avoids the use of pumps
for air feeding. A pump is required only to move
the nutrient solution from the bottom to the top
of the bioreactor [83]. The low mobility of the
cells in the PSBR has been also indicated as a
promising solution to contain contamination,
because when that appears, it remains confined
to a limited area.
Very high productivities have been reached
by using PSBRs. In fact, for Scenedesmus obliquus
 3. Innovative photobioreactors 297

FIGURE 16.4 Schematic representation of the porous substrate bioreactor (PSBR) configuration.

and Arthrospira platensis, biomass productivities
until 80 and 60 g/m2 per day were achieved,
respectively [84,85]. The same works measured
very high photosynthetic efficiency too, with
values greater than 10% [85]. These values are
significantly higher with respect to the values
typically achieved by using conventional configu-
rations pilot plants, which are between 5 and
20 g/m2 per day. However, these studies were
carried out by maintaining the reactors under a
closed chamber in which CO2 concentration in
the air was strongly enriched (until 2%e5%)
with respect to environmental air (0.04%). In
contrast, by using the environmental CO2 concen-
tration, a productivity value of 7 g/m2 per day
was obtained; this value is comparable with those
obtained from conventional configurations [86].
These data indicate that gas exchange from envi-
ronmental air to the biofilm is the limiting factor
for microalgae growth in PSBRs. Some other rele-
vant factors that can strongly affect the biomass
productivity are light dilution generated by the
design of PSBRs and biomass loss by respiration
in the deep part of the biofilm [82]. More data
are required from outdoor pilot plants operated
for different seasons to carry out detailed cost
analysis comparisons.
3.3 Other innovative solutions to
improve phototrophic cultivation
Besides the configurations described in Sec-
tions 3.1 and 3.2, there are some other systems
that are being investigated to improve microal-
gae cultivation sustainability. One recent
example is given by the liquid foam-bed photo-
bioreactor [87]. This kind of reactor is based on
the idea of growing microalgae on the surface

IV. Selected examples and case history

298 16. New strategies enhancing feasibility of microalgal cultivations
of foam bubbles. The advantages are similar to
those of biofilm reactors. Microalgae are directly
exposed to air, so they can grow faster by
exploiting the low light path, and harvesting
can be more efficient thanks to the higher
biomass concentration. Microalgae are inocu-
lated on a suspension containing a surfactant be-
sides nutrients. The suspension is fed at the
bottom of a photobioreactor together with a
gas flux. By passing through a gas distributor
plate, the foam is generated, filling the reactor
to the top. Then the foam goes from the top of
the reactor to a foam breaker (made, for example,
of a fixed bed filled with plastic beads), and the
medium is recirculated by a pump. A key factor
that can influence the performance of the reactor
is given by the surfactant. Ideally, it should have
good foaming properties (e.g., foam stability and
bubble size), it should be nonbiodegradable,
chemically stable, biocompatible with algae,
cheap, and it should allow a good repartition
of microalgae to the foam phase. By comparing
several surfactants, Pluronic F68 and P84 were
found to be the best after the evaluation of
different criteria [88]. When bovine serum albu-
min (BSA) was used as surfactant, a time of oper-
ativity of only 8 h was maintained, due to the
instability of BSA [87]. In a first study, a lower
growth rate with respect to conventional config-
urations was obtained due to the dark volume
of the culture, which accounted for about
30% of total volume [87]. However, with an
improved version of the liquid foam-bed photo-
bioreactor, in which Pluronic F68 was used as
surfactant and in which liquid recirculation
was introduced, higher growth performances
were achieved. Chlorella vulgaris and
Chlorella sorokiniana were cultivated in contin-
uous mode, for 500 h, maintaining biomass con-
centration around 20e25 g/L and reaching
growth rate values to 0.1 h
1 [89].
To reduce water and energy consumption for
temperature control in a photobioreactor, a so-
lar control infrared blocking film (purchased
by 3M) was tested [90]. The film prevented
IV. Selected examples and case history
infrared light penetration, reducing maximum
temperature achieved by 33% and increasing
by 52% and 64% biomass production and
phycocyanin content, respectively, with respect
to reactors without any temperature control
system. The growth performances were compa-
rable with respect to those achieved by using
conventional temperature control systems as
water jacket.
Other innovative materials can be exploited to
obtain reactor surfaces able to convert a part of
the inactive region of the solar spectrum to
wavelengths that can be actively used by photo-
synthesis [8].
Some authors investigated the efficacy of
stacked waveguide reactors, which have micro-
patterned waveguide surfaces. These reactors
exploit evanescent fields near the surface to
enhance microalgae growth, which was
increased two- to fourfold [91,92].
In some studies, the enhancement of microal-
gae productivity by fighting grazers was inves-
tigated. Grazers are organisms that can
contaminate photoautotrophic microalgae cul-
tures (especially open ponds) by decreasing
biomass productivity, generating culture
crashes. Following the same approach used to
fight pests in terrestrial cultivations, several
chemical compounds were studied for their ef-
fects on grazers and on microalgae; among
these, 21 chemicals effective against grazers
and compatible with Chlorella sp. were found.
Benzalkonium chloride was tested in open
pond, proving its ability in killing grazers and
preventing negative effects on microalgae
growth [93].
4. Wastewaters utilization as a source of
nutrients
The integration of microalgae cultivation with
wastewater treatment is a promising approach
that can improve the feasibility of microalgae
biomass production. The main features that
 4. Wastewaters utilization as a source of nutrients
may potentially increase the feasibility are as
follows:
- Wastewater addition can reduce nutrient
consumption, which is an issue both for
economic (cost for nutrient purchase) and
environmental reasons (resource depletion).
- Wastewater treatment can enhance economic
sustainability by making the pollution
treatment an added value of the process.
- Wastewater treatment can enhance
environmental sustainability by reducing
pollutants spreading in the environment.
- Organic molecules in wastewaters can work
as substrate for mixotrophic and
heterotrophic cultivations, which can
increase remarkably biomass productivity
and biomass concentration with respect to
photoautotrophic conditions.
The treatment of several wastewaters has
been tested for integration with microalgae culti-
vation. These include municipal wastewaters
[94,95], manure wastewaters [96e98], digestate
from anaerobic treatments [99,100], meat pro-
cessing wastewater [101], cheese whey
[102,103], olive mill wastewater [104,105], and
textile wastewaters [106].
The aim of this section is to describe the latest
innovations and the potentials of wastewater uti-
lization in microalgae processes. In Subsection
4.1 the potential of wastewaters in nutrients sup-
plementation is described, while in Subsection
4.2 the performances of microalgae processes in
pollutants removal from wastewater are
TABLE 16.2 Empirical formula for microalgae biomass.
Microalgae Empirical formula
- CH2.4ON0.15P0.009
T. obliquus CH1.9O0.6N0.13P0.012K0.01Mg0.006S0.003Ca0.0004
C. vulgaris CH2O0.5N0.17P0.014K0.01Mg0.003
- CH2.5ON0.15P0.009
IV. Selected examples and case history
299
reported. Subsequently, in Section 4.3, the
room of improvement that can be achieved by
the exploitation of organic substrates from
wastewater by microalgae heterotrophic meta-
bolism is outlined.
4.1 Wastewater as a source of nutrients
for microalgae growth
Photosynthesis is the metabolic pathway by
which microalgae convert H2O and CO2 to
organic compounds such as sugars (CH2O)n.
However, H2O and CO2 are not the only nutri-
ents required for microalgae growth. Other nu-
trients are required to supply elements such as
N, P, S, Mg, and others, which are essential for
the production of new microalgae cells [2]. The
empirical formula for microalgae cells has been
calculated in some different works, and the
values determined are summarized in
Table 16.2.
For a sustainable, large-scale production of
microalgae, it is essential that all the nutrients
are used in a sustainable way to minimize
resource depletion from the earth. It has been
calculated that if the biodiesel required to sup-
port the European transportation market is pro-
duced entirely by microalgae, the amount of P
and N required would be twice the amount
that is presently produced as fertilizer in Europe
[9]. Consequently, recycling nutrients is essential
for a sustainable production of microalgae.
Several studies have been conducted with the
aim of studying the recycling of nutrients
Source
[107]
[108]
[43]
[109]
300 16. New strategies enhancing feasibility of microalgal cultivations
coming from wastewaters in place of synthetic
nutrients. In a large part of the studies carried
out, the wastewaters were diluted with distilled
water, tap water, or synthetic culture medium.
That is because 100% of wastewaters often
generate issues given by the high concentration
of substances limiting microalgae growth by
reducing light penetration or by their antimicro-
bial effects (such as the case of phenols and NHþ 4)
[110,111]. Municipal wastewater (MW) is one of
the most studied as a source of nutrients to sup-
port microalgae growth. MW has an N/P w 3
[112], lower than the ratio typically required for
microalgae growth, which is w15 (Table 16.2).
Therefore, nitrogen is the limiting nutrient in
such wastewaters. Nitrogen concentration
ranges between 10 and 100 mg/L in MW. Such
a concentration can support a biomass produc-
tion to 0.1e1 g/L before becoming limiting
[113]. Generally, phosphorus is not a limiting
nutrient; however, it should be considered that
at some Ca2þ concentrations, which may be
found in tap water, phosphorus can becomes
easily limiting because of phosphates precipita-
tion [114].
Digestate effluents originating from anaerobic
treatments have higher nutrients concentrations
with respect to MW. Nitrogen can vary in the
range of 100e3500 mg/L and phosphorus be-
tween 20 and 400 mg/L. The N/P ratio is a bit
more favorable too, about 3.6e4.3 [112]. Howev-
er, nitrogen is present mainly as NHþ 4 , which has
toxic effects for microalgae at too high concentra-
tions [115]. There is not a fixed threshold limit for
NHþ 4 concentration because its effect can vary
depending on the microalgae strain and cultiva-
tion conditions. However, the reported values
over which negative effects were detected
ranged between 50 and 300 mg/L [116]. For
this latter reason, digestates need to be diluted
to be used as a nutrients source for microalgae
cultivation. Moreover, it should be considered
that microalgae alkalinize the pH by the photo-
synthetic activity. Therefore, if pH is not
controlled the large part of the nitrogen could
IV. Selected examples and case history
be lost by means of NH3 evaporation
(NHþ þ
4 þ H2O 4 NH3 þ H3O , pKa ¼ 9.25).
Olive mill wastewater (OMW) is produced
during the milling of olives to produce olive
oil. OMW is characterized by a high content of
phosphate and other mineral salts, but it has a
low content of nitrogen available for microalgae
growth [114,117]. To support microalgae
growth, OMW typically requires an addition of
nitrogen [118,119] that can be made also by mix-
ing OMW with other wastewaters like urban
wastewaters [120].
Cheese whey (CW), which is produced from
cheese production plants, is rich in nitrogen
and phosphorus. These nutrients are both in
organic (mainly proteins) and inorganic forms
(P-PO3
4 and NeNHþ 4 ). Proteins are generally
removed from whey by filtrations to isolate
whey proteins, which have high added value
[103]. Inorganic nitrogen and inorganic phos-
phorus range between 65 and 160 mg/L and
300e400 mg/L, respectively [121]. The N/P ra-
tio is w0.3, about 10-fold lower than MW and
digestate effluents. It has been proven that CW
can be used to sustain microalgae growth by
replacing the addition of inorganic salts; howev-
er, only a few strains are able to use lactose as a
substrate [103], which is mainly responsible for
the high COD and BOD5 content in CW [121].
The organic substrates that can be found in
wastewaters (generally measured as COD,
BOD5, or TOC) can furnish further nutrients for
microalgae growth. They can be a source of en-
ergy and mass (C, H, O, N, S, P) for microalgae
metabolism. The exploitation of this latter class
of nutrients is described in detail in Section 4.3.
Recently, the interest about the possibility to
recycle directly nutrients from microalgae
biomass is growing too. When the target prod-
ucts from microalgae are, for example, TAGs
and starch, N and P are not included in the prod-
ucts; consequently, their recycling may be
possible in an efficient process. Microalgae
biomass can be used as substrate for anaerobic
digestion, allowing one to recover about 40 g of
 4. Wastewaters utilization as a source of nutrients
N per kg of dry biomass (69%e86%) and 3.8 g of
P per kg of dry biomass (9%e49%) [122]. Hydro-
thermal liquefaction can be also used to recycle
nutrients from microalgae biomass, allowing
one to recover about 26 g of N per kg of dry
biomass (15%e84%) and 6.8 g of P per kg of
dry biomass (20%e85%) [122].
4.2 Wastewater treatment by microalgae
Microalgae cultivation can reduce the
polluting load of wastewaters by removing
organic and inorganic molecules. Such removal
can be obtained by several mechanisms, such
as enzymatic conversion to other molecules,
storage inside cells (bioaccumulation), adsorp-
tion on cell surface (bioadsorption), variation of
chemicalephysical parameters (e.g., pH, O2 con-
centration), and action of bacteria that live in as-
sociation with microalgae.
For example, inorganic N and P can be accu-
mulated inside cells and then used as a substrate
by the enzymes to produce proteins, mem-
branes, and nucleic acids. Heavy metals can be
bioaccumulated and/or adsorbed [123,124].
COD can be reduced by converting organic mol-
ecules to CO2 and H2O (mineralization) or to
other organic molecules with a lower oxygen de-
mand. The pH increase induced by photosyn-
thetic activity can boost phosphates and metals
removal by precipitation and adsorption
[114,123,125].
From MWs, high phosphorus and nitrogen
removal, between 80% and 100%, has been usu-
ally obtained. These results were achieved by us-
ing several microalgae species (e.g., Chlorella sp.
and Scenedesmus sp.) and several reactor config-
urations (open ponds, photobioreactors, immo-
bilized cells) [126e129]. COD, TOC, and BOD
removal has been also obtained at comparable
high values, close to 100%. However, for these
latter parameters, which are linked to the organic
compounds, a main role is played by the bacteria
communities that live in symbiosis with
IV. Selected examples and case history
301
microalgae [130]. Bacteria contamination is a
limited issue for this kind of wastewater because
the initial COD concentration is quite low (be-
tween 100 and 500 mg/L) [131]. Several studies
have been conducted to exploit a synergic inter-
action between microalgae and bacteria. It
should be considered that in conventional waste-
water treatment plants, the aeration required to
support aerobic bacteria can account for 45%
e75% of the energy consumption [132]. By using
a mixed culture of microalgae and bacteria,
microalgae can produce O2 by photosynthesis,
reducing the energy supply for aeration and
furnishing at the same time O2 for COD reduc-
tion by bacteria. Bacteria oxidation of organic
molecules converts the O2 to CO2, which can
be used again by microalgae metabolism in a
closed cycle [133,134].
Digestate produced from anaerobic fermenta-
tion can be also efficiently treated by microalgae
culture to reduce the concentration of pollutants.
Removal efficiencies of N and P to 70%e100%
have been obtained for digestate produced
from swine manure, dairy manure, MWs,
poultry wastewaters, and others [116]. Because
of the higher pollutant concentration in anaer-
obic digestates with respect to MWs, they often
require a pretreatment before microalgae culti-
vation. Generally, the pretreatments carried out
are dilution, filtration, precipitation, steriliza-
tion, and centrifugation [116]. The removal of
COD can achieve efficiencies to 50%e90%. How-
ever COD initial concentration has higher values
with respect to MWs, generating a relevant issue
by biologic contamination [116]. In anaerobic
digestate, the high content of metal ions (Cu2þ,
Pb2þ, Zn2þ, Co2þ, and others) is also a relevant
issue. Microalgae growth can reduce signifi-
cantly the concentration of metals and heavy
metals; this has been proven, for example, for
Zn2þ and Cd2þ [135]. However the large part
of the studies carried out on this field did not
evaluate this aspect.
The direct cultivation of microalgae in agro-
industrial wastewaters such as CW has been
302 16. New strategies enhancing feasibility of microalgal cultivations
proven, allowing lactose removal to 64% [102]
and nitrogen removal to 100% [103]. However,
lactose can be used as substrate only by some
microalgae strains [103]. Moreover, in the re-
ported studies, all the experiments were carried
out under fully sterilized conditions, which are
hardly applicable to large-scale cultivation.
Microalgae cultivation on diluted OMW was
efficient in phenols removal to 50%e60%
[105,110,114], COD reduction to 30%e40%
[118] and phosphates removal to 91% [114].
4.3 Enhanced microalgae growth by
exploiting organic substrates from
wastewaters
The large part of wastewaters contains signifi-
cant amounts of organic substrates. Many micro-
algae can use such organic substrates, by means
of a heterotrophic metabolism, as a source of car-
bon and energy, in addition to light and CO2
exploited by the photoautotrophic metabolism.
When the heterotrophic metabolism is operated
concurrently with photoautotrophic meta-
bolism, the microalgae metabolism is called mix-
otrophic (heterotrophic þ photoautotrophic).
This further source of energy and carbon gives
a room of improvement for microalgae biomass
production processes. In this subsection, such
possibility of improvement is described.
4.3.1 Microalgae growth on organic
substrates
The organic molecules that are present in
wastewaters have the possibility to boost micro-
algae productivity by feeding their heterotrophic
metabolism. Heterotrophic metabolism is inde-
pendent of light penetration inside reactors;
consequently, it could allow achieving high
biomass concentration and biomass productivity
by cultivating microalgae in reactors with a low
S/V ratio.
Microalgae can use a large variety of organic
molecules as source of carbon and energy;
IV. Selected examples and case history
however, this ability can vary from one strain
to another strain. Glucose is likely the most
tested substrate, and it has been used for the
cultivation of a large number of different strains
including Chlorella protothecoides, Chlorella
regularis, Chlorella vulgaris, Chromochloris
zofingiensis, Crypthecodinium cohnii, Dunaliella sp.,
Euglena gracilis, Galdieria sulphuraria, Scenedesmus
sp., and many others [43,136]. Other sugars have
been studied less. Sucrose is generally not
useable directly by microalgae because of the
absence of invertase [137]. Lactose can be used
only by a few strains [102,103]. Glycerol and
acetate have been widely used to support
the growth of several microalgae species
[43,136,138]. Other substrates that have been
also used for microalgae cultivations are some
amino acids (such as glutamate, aspartate, aspar-
agine), butyric acid, ethanol, galactose, mannose,
and fructose [43,136,139].
When cultivated under a heterotrophic
regime, microalgae have yield factors that usu-
ally range from 0.3 to 0.7 g of dry weight
biomass per gram of organic substrate [43]. The
yield factor varies depending on the strain, the
kind of organic substrate, and the cultivation
conditions. Considering that several wastewa-
ters have concentrations of organic compounds
to w50 g/L, it can be deduced that more than
30 g/L of microalgae biomass may be obtained
by adding wastewater as a nutrient source.
Moreover, because of the independence of the
heterotrophic metabolism from the light supply,
it should be possible to obtain also high biomass
productivities by working with low-cost config-
urations (low S/V ratio).
However, similar improvements in microal-
gae biomass production and productivity have
been registered only for microalgae cultivated
in fully sterilized conditions [43]. That is because,
without an accurate sterilization process and
without an axenic microalgae culture, the
organic substrate is used more efficiently by
the contaminant bacteria [116,140]. Bacteria can
easily outclass microalgae growth in the
 4. Wastewaters utilization as a source of nutrients
presence of high substrate concentration because
their maximum growth rate (w20 d
1) is about
one order of magnitude higher than microalgae
(w2 d
1).
Several studies have been also conducted by
using unsterilized wastewaters, namely by
working with a consortia of bacteria and micro-
algae. However, in these cases the attention
was mainly focused on wastewater pollutants
removal, and the initial COD concentrations
were generally low (<1 g/L) because of the
kind of wastewater used (e.g., MWs) or because
of wastewater dilution [129,134,141,142]. In
these conditions, contamination is strongly
limited by the low COD, but at the same time,
microalgae growth is mainly phototrophic,
without any relevant advantage given by the
organic substances.
Only in some cases, unsterilized wastewater
was used to exploit directly the advantage given
by the supplemented organic substrates. For
example, OMW was added in a two-stage photo-
trophic-heterotrophic cultivation process. In
such a process, microalgae were first cultivated
in a photoautotrophic regime, to reach nitrogen
depletion, and then the wastewater was added
generating biomass production and productivity
comparable to the photoautotrophic control
[19,105]. Although there was not an improve-
ment in biomass growth with respect to the con-
trol, the possibility to obtain a comparable
growth in a reactor with lower S/V ratio was
already a gain.
The use of wastewaters as a source of organic
substrates to improve microalgae biomass pro-
ductivity has not yet been proven in a way that
is technically promising. The issue of bacterial
contamination to nonaxenic conditions is the
main limit that needs to be solved.
4.3.2 Innovative strategies for organic
substrate addition
Some innovative strategies have been investi-
gated with the aim to control bacteria growth in
nonaxenic microalgae cultivations. A factor that
IV. Selected examples and case history
303
can be exploited to favoring microalgae growth
over bacteria is the different ability to store nutri-
ents [109]. Microalgae growth is generally
described by a Droop model [113,143], which de-
scribes the ability of microalgae to store nitrogen
and to grow in N-starvation condition by using
the internal nitrogen quota. Instead, a large
part of bacteria show a reduced ability to store
nutrients, and their growth is generally
described by a Monod model, which means
that growth is stopped as soon as nutrients are
depleted in the cultivation media [140]. This dif-
ference was exploited by a mixotrophic cultiva-
tion in which glucose and nitrate were added
alternatively [144]. In the described alternate
feeding strategy, a fixed amount of glucose and
nitrate were added separately and alternatively
every 2 days, under mixotrophic regime. The
strategy was compared with a mixotrophic con-
trol in which glucose and nitrate were added
simultaneously every 2 days. The authors found
that with the alternate strategy, bacterial
contamination was strongly reduced. During
the alternate strategy, there was an alternance
of low glucose/high nitrate concentration condi-
tion and low nitrate/high glucose concentration
condition. Microalgae could grow under the low
nitrate concentration condition (N-starvation) by
exploiting the internal nitrogen quota, and under
the low glucose concentration condition by using
the extra inorganic carbon (CO2) by means of
photosynthesis.
The efficacy of a similar strategy was assessed
also in heterotrophic condition [177]. In this
latter case, the ability of microalgae to store
organic substrates was exploited in addition to
the nitrogen storage. In fact, in heterotrophic
condition, the growth of microalgae under high
nitrate/low glucose concentration condition
was only possible by exploiting the microalgae
ability to use solely the internal quota of organic
molecules stored as a source of energy (mainly
starch and lipids). In this strategy, named
uncoupled heterotrophic strategy (UHS),
glucose and nitrate were added alternatively,
304 16. New strategies enhancing feasibility of microalgal cultivations
by tailoring their initial concentrations as a
function of the biomass concentration and of
the internal quota of organic compounds stored.
Moreover, the time between nitrate addition and
the subsequent glucose addition was tailored as
a function of the consumption kinetic. These ad-
justments on glucose and nitrate addition
avoided low biomass growth or high bacterial
contamination given by underestimation or
overestimation of the substrate consumption in
every phase.
With the UHS strategy, a strong reduction in
bacteria concentration was achieved, and at the
same time, 6 g/L per day and 26 g/L of biomass
productivity and biomass concentration were
achieved, respectively.
5. Strain improvement
Microalgaecultivationcanbeseenasanewkindof
agriculture. The conventional agriculture that we
knowistheresultofthousandsofyearsofimprove-
ments. These changes have not been directed only
byagronomicpractices,butakeyandfundamental
rolewasalsoplayedbythegeneticimprovementof
the species cultivated [145]. The reason at the
base of this latter fact is that plants in nature
are the results of millions of years of evolution,
but this evolution was mainly directed by their
fitness, which means by their ability to repro-
duce themselves in the environmental conditions
in which they live and in the new environmental
conditions with which they had to adapt during
the years. Consequently, the parameters that
have been improved in plants living in nature
were, for example, the production of resistant
seeds, leaves, and stems to face herbivorous
and competitors. By means of agriculture,
several species were genetically improved to-
ward the characteristics requested for cultiva-
tion. The natural environment was replaced by
the agricultural field, a more comfortable
environment, generated by the human work, in
which plants could grow using more energy in
IV. Selected examples and case history
producing bigger fruits and seeds instead of
in defense against the environment. The differ-
ences between maize and its ancestor teosinte
(Zea mays ssp. mexicana) can be considered an
example [146]. The large part of the current agri-
cultural production derives from the genetic im-
provements obtained by the “green revolution”
[145]. Analogous to what was done with terres-
trial plants, the future of the cultivation of micro-
algae will be likely strongly related to the genetic
improvements. A “green revolution” is expected
to be a key factor to improve the feasibility of
microalgae cultivation processes [147].
In the following subsections, the main meta-
bolic pathways that have been manipulated in
microalgae by means of genetic modification
are described.
5.1 Improvement of photosynthetic
efficiency
Photosynthetic efficiency of microalgae is
measured as the ratio between energy supplied
to microalgae by light illumination and the en-
ergy content of the microalgae biomass pro-
duced by photosynthesis. It should be
considered that from the atmosphere to the
ground, only a fraction of the light energy, esti-
mated between 10% and 34%, reaches the
external surface of the reactors [8]. The energy
is lost due to atmospheric scattering, weather
conditions, latitude, reactor orientation, and the
limited photosynthetic active region. Then, other
losses can be given by light saturation of cells
and by energy consumption of the microalgae
metabolism. Considering only the incident light
on the reactor surface, the typical photosynthetic
efficiency registered is around 0.1%e10% and
the theoretical maximum value is w12% [8]. A
factor that can reduce photosynthetic efficiency
is photoinhibition, which can occur when micro-
algae are exposed to too high a flux of photons.
In this condition, the rate of photon absorption
by light harvesting antenna becomes higher
 5. Strain improvement
than the rate at which photons are utilized by
metabolism. The excess of photons (to 80%) is
lost as heat, damaging the photosystems [148].
To overcome this issue, some genetically modi-
fied strains of Chlamydomonas reinhardtii have
been produced, which have a truncated light
harvesting antenna [149e151]. These strains
have reduced the amount of chlorophyll for light
harvesting, making them less sensitive to photo-
inhibition. Moreover, their reduced light adsorp-
tion per unit cell reduces the self-shading effect
inside photobioreactors too.
5.2 Improvement in target compound
accumulation
As well as maize that was genetically
improved to accumulate more starch in bigger
cobs with respect to its wild ancestor, the same
approach can be followed to induce microalgae
to accumulate higher amounts of target com-
pounds. In this direction, several microalgae
strains able to accumulate higher amounts of
TAG have been obtained by inhibiting the syn-
thesis of starch (starchless mutants). Most
microalgae accumulate organic carbon during
N-starvation both as TAG and as starch, which
are two competitive pathways. Limiting the starch
pathway, the flux of carbon assimilation is directed
mainly to TAG synthesis. Starchless mutant strains
have been obtained for Chlorella pyrenoidosa,
Chlamydomonas reinhardtii, and
Tetradesmus obliquus, showing a higher level of
TAG accumulation [152,153].
Other pathways can be also changed to
improve lipid accumulation; for example, in
Phaeodactylum tricornutum the neutral lipid
content was increased by 82% by inactivating
pyruvate dehydrogenase kinase [154]. For the
same species, an increase in lipid content up
to 2.5-fold was obtained with a transgenic
strain that overexpressed malic enzyme [155].
Chlamydomonas reinhardtii mutants with a
more than twice improved starch accumulation
IV. Selected examples and case history
305
ability, with respect to wild-type control, were
obtained by using gamma irradiation [156]. For
the same species, a mutant deficient for a dual-
specificity tyrosine phosphorylation-regulated
kinase showed a higher level of starch accumula-
tion with respect to wild type [157].
Some researchers attempted to enhance
accumulation of pigments too. By expressing
a phytoene synthase gene (CzPSY) from
Chlorella zofingiensis to Chlamydomonas reinhardtii,
2.0- and 2.2-fold increases were obtained for vio-
laxanthin and lutein accumulation, respectively
[158].
5.3 Improvement in strain resistance to
adverse conditions
The environment inside bioreactors is
controlled within certain values to maintain the
main environmental parameters around the
optimal values. However, two things should be
considered: (1) it is impossible to control every
parameter, and there will be invariably some pa-
rameters that may vary until producing stressful
conditions for microalgae. (2) Every control sys-
tem has a cost that affects the economic sustain-
ability of the cultivation process.
Therefore, the ability of microalgae strains to
resist to the adverse/nonoptimal conditions
encountered during cultivation is a key factor
to consider in strain selection and improvement.
Without any control system, temperature can
vary inside a photobioreactor in the range of
below 0e50 C, affecting strongly microalgae
growth. A lot of energy is required to cool
down water temperature during warm seasons
and to raise water temperature during winter
seasons [159]. For this reason, many efforts
have been made looking for strains more tolerant
to temperature fluctuations. A mutant of
Chlorella sp., obtained by chemical mutagenesis,
showed higher tolerance to high temperature
(to 45 C) with respect to wild type [160].
Similarly, a thermotolerant mutant of
306 16. New strategies enhancing feasibility of microalgal cultivations
Chlorella pyrenoidosa (NCIM 2738) was cultivated
under subtropical outdoor conditions corre-
sponding to 43e45 C during the day and
27e30 C during the night [161]. Five mutant
strains of Arthrospira were obtained by ethyl
methanesulfonate mutagenesis. These strains
grew better than the wild-type strain at low tem-
perature, by achieving biomass production
10-fold higher than wild type at 16 and 18 C
[162]. Improvement of Chlamydomonas growth
was obtained at 10 C by using a diploid strain
[163] and at 4 C by using AMP deaminase sup-
pression [164]. The overexpression of hspA and
osmotin genes in Synechococcus elongatus
induced a coupled improved tolerance to high
temperature (45 C), high salt concentration
(3.5%), and high illumination (300 mmol/m2 per
second) [165].
Another important aspect is CO2 concentra-
tion. CO2 is often a limiting nutrient in photo-
bioreactors; consequently, to obtain higher
biomass productivities, the photobioreactors
could be supplied with higher CO2 concentra-
tions in the feeding gas. However, CO2 concen-
trations higher than 10% can have toxic affects
on microalgae [166,167]. For this reason,
different works have been conducted to isolate
microalgae strains more tolerant to high CO2
levels. Strains able to tolerate CO2 concentra-
tions to 20%e30% have been obtained [167,168].
Apart from the main environmental parame-
ters considered during microalgae cultivation,
several pollutants present in liquid and air
streams feeding the reactors can affect microal-
gae growth too. Utilization of flue gas in place
of pure CO2 to supply photobioreactors can
potentially reduce production costs. However,
flue gas contains other compounds beyond
CO2, such as NOx and SOx, that can limit micro-
algae growth [169]. A Chlorella sp. strain
tolerant to flue gas was obtained by 46 cycles
of adaptive evolution, showing the same
growth on synthetic flue gas compared to pure
CO2 [170]. Several microalgae strains resistant
to pollutants found in different wastewaters
IV. Selected examples and case history
have been selected, for example, microalgae
strains more tolerant to phenols [171], to pesti-
cides [93,172], to heavy metals [173], to
extremely acid pH [174,175], and to high
salinity [165,176].
6. Conclusions and future trends
It can be concluded that although the cost of
microalgae biomass production is limiting for a
large part of the possible industrial applications,
this technology has a large room for improve-
ment. The future trends expected for the scienti-
fic and industrial research in microalgae
processes development are as follows:
(a) the development of new reactors by which
energy and cost associated with the
cultivation of microalgae can be reduced;
(b) the development of new microalgae strains
for a new “green revolution” by which the
efficiency of target compounds production in
the reactor environment can be maximized;
(c) the development of cultivation processes
integrated with wastewater treatment for the
exploitation of organic substrates and for
nutrient recycling;
(d) the development of tailored systems to
control biologic contaminants;
(e) the development of biorefinery facilities by
which several end products can be produced
starting from microalgae biomass.
List of abbreviations and acronyms
AMP Adenosine monophosphate
BOD5 Biochemical oxygen demand (measured after 5 days
of incubation)
BSA Bovine serum albumin
COD Chemical oxygen demand
CW Cheese whey
DHA Docosahexaenoic acid
MW Municipal wastewater
OMW Olive mil wastewater
PSBR Porous substrate bioreactors
 References
TAGs Triacylglycerols
UFP Ultrathin flat photobioreactor
[13]
References
[1] E. Walz, S. Energy, E. Walz, Biotech’s green gold? Na-
ture Biotechnology 27 (2009) 15e18, https://doi.org/ [14]
10.1038/nbt0109-15.
[2] G. Markou, D. Vandamme, K. Muylaert, Microalgal
and cyanobacterial cultivation: the supply of
nutrients, Water Research 65 (2014) 186e202,
https://doi.org/10.1016/j.watres.2014.07.025. [15]
[3] P. Spolaore, C. Joannis-Cassan, E. Duran, A. Isambert,
Commercial applications of microalgae, Journal of
Bioscience and Bioengineering 101 (2006) 87e96,
https://doi.org/10.1263/jbb.101.87.
[4] J.A. Garrido-Cardenas, F. Manzano-Agugliaro,
F.G. Acien-Fernandez, E. Molina-Grima, Microalgae [16]
research worldwide, Algal Research 35 (2018)
50e60, https://doi.org/10.1016/j.algal.2018.08.005.
[5] K.M. Weyer, D.R. Bush, A. Darzins, B.D. Willson,
Theoretical maximum algal oil production, Bioenergy
Research 3 (2010) 204e213, https://doi.org/10.1007/ [17]
s12155-009-9046-x.
[6] X.G. Zhu, S.P. Long, D.R. Ort, What is the maximum
efficiency with which photosynthesis can convert so-
lar energy into biomass? Current Opinion in Biotech-
nology 19 (2008) 153e159, https://doi.org/10.1016/ [18]
j.copbio.2008.02.004.
[7] M.R. Tredici, Photobiology of microalgae mass cul-
tures: understanding the tools for the next green
revolution, Biofuels 1 (2010) 143e162, https://
doi.org/10.4155/bfs.09.10.
[8] M.D. Ooms, C.T. Dinh, E.H. Sargent, D. Sinton, [19]
Photon management for augmented photosynthesis,
Nature Communications 7 (2016) 12699, https://
doi.org/10.1038/ncomms12699.
[9] R.H. Wijffels, M.J. Barbosa, An outlook on microalgal
biofuels, Science 329 (2010) 796e799, https://
doi.org/10.1126/science.1189003. [20]
[10] K.S. Dhugga, Maize biomass yield and composition
for biofuels, Crop Science 47 (2007) 2211e2227,
https://doi.org/10.2135/cropsci2007.05.0299.
[11] M.R. Tredici, L. Rodolfi, N. Biondi, N. Bassi,
G. Sampietro, Techno-economic analysis of microal-
gal biomass production in a 1-ha Green Wall Panel [21]
(GWPÒ) plant, Algal Research 19 (2016) 253e263,
https://doi.org/10.1016/j.algal.2016.09.005.
[12] M. Vigani, C. Parisi, E. Rodríguez-Cerezo,
M.J. Barbosa, L. Sijtsma, M. Ploeg, C. Enzing, Food [22]
and feed products from micro-algae: market
IV. Selected examples and case history
307
opportunities and challenges for the EU, Trends in
Food Science and Technology 42 (2015) 81e92,
https://doi.org/10.1016/j.tifs.2014.12.004.
J.M. MacDonald, P. Korb, R.A. Hoppe, Farm Size and
the Organization of U.S. Crop Farming, ERR-152,
2013. http://www.ers.usda.gov/media/1156726/
err152.pdf.
F.G. Acien, J.M. Fernandez, J.J. Magan, E. Molina, Pro-
duction cost of a real microalgae production plant and
strategies to reduce it, Biotechnology Advances 30
(2012) 1344e1353, https://doi.org/10.1016/
j.biotechadv.2012.02.005.
J. Ruiz, G. Olivieri, J. de Vree, R. Bosma, P. Willems,
J.H. Reith, M.H.M. Eppink, D.M.M. Kleinegris,
R.H. Wijffels, M.J. Barbosa, Towards industrial prod-
ucts from microalgae, Energy and Environmental Sci-
ence 9 (2016) 3036e3043, https://doi.org/10.1039/
C6EE01493C.
N.H. Norsker, M.J. Barbosa, M.H. Vermuë,
R.H. Wijffels, Microalgal production e a close look at
the economics, Biotechnology Advances 29 (2011)
24e27, https://doi.org/10.1016/
j.biotechadv.2010.08.005.
C.Y. Chen, X.Q. Zhao, H.W. Yen, S.H. Ho, C.L. Cheng,
D.J. Lee, F.W. Bai, J.S. Chang, Microalgae-based car-
bohydrates for biofuel production, Biochemical Engi-
neering Journal 78 (2013) 1e10, https://doi.org/
10.1016/j.bej.2013.03.006.
S. Pierucci, J.J. Klemes, L. Piazza, S. Bakalis, I. Gifuni,
G. Olivieri, I.R. Krauss, G. D’errico, A. Pollio,
A. Marzocchella, Microalgae as new sources of starch:
isolation and characterization of microalgal starch
granules, Chemical Engineering Transactions (2017),
https://doi.org/10.3303/CET1757238.
F. Di Caprio, A. Visca, P. Altimari, L. Toro,
B. Masciocchi, G. Iaquaniello, F. Pagnanelli, Two stage
process of microalgae cultivation for starch and
carotenoid production, Chemical Engineering Trans-
actions 49 (2016) 415e420, https://doi.org/10.3303/
CET1649070.
C. Schulze, M. Wetzel, J. Reinhardt, M. Schmidt,
L. Felten, S. Mundt, Screening of microalgae for primary
metabolites including b-glucans and the influence of ni-
trate starvation and irradiance on b-glucan production,
Journal of Applied Phycology 28 (2016) 2719e2725,
https://doi.org/10.1007/s10811-016-0812-9.
H.M.D. Wang, C.C. Chen, P. Huynh, J.S. Chang,
Exploring the potential of using algae in cosmetics,
Bioresource Technology 184 (2015) 355e362,
https://doi.org/10.1016/j.biortech.2014.12.001.
T. Chiong, C. Acquah, S.Y. Lau, E.H. Khor,
M.K. Danquah, Microalgal-based protein by-products:
308 16. New strategies enhancing feasibility of microalgal cultivations
extraction, purification, and applications, in: Protein
Byprod. Transform. From Environ. Burd. Into Value-
Added Prod., 2016, pp. 213e234, https://doi.org/
10.1016/B978-0-12-802391-4.00012-4.
[23] M. Hayes, H. Skomedal, K. Skjånes, H. Mazur-
Marzec, A. Toru nska-Sitarz, M. Catala, M. Isleten
Hosoglu, M. García-Vaquero, Microalgal proteins for
feed, food and health, in: Microalgae-Based Biofuels
Bioprod. From Feed. Cultiv. to End-Products, 2017,
pp. 347e368, https://doi.org/10.1016/B978-0-08-
101023-5.00015-7.
[24] M.A. Zeller, R. Hunt, A. Jones, S. Sharma, Bioplastics
and their thermoplastic blends from Spirulina and
Chlorella microalgae, Journal of Applied Polymer Sci-
ence 130 (2013) 3263e3275, https://doi.org/10.1002/
app.39559.
[25] S. Kumar, E. Hablot, J.L.G. Moscoso, W. Obeid,
P.G. Hatcher, B.M. Duquette, D. Graiver,
R. Narayan, V. Balan, Polyurethanes preparation us-
ing proteins obtained from microalgae, Journal of Ma-
terials Science 49 (2014) 7824e7833, https://doi.org/
10.1007/s10853-014-8493-8.
[26] C.A. Ovando, J.C. de Carvalho, G. Vinícius de Melo
Pereira, P. Jacques, V.T. Soccol, C.R. Soccol, Func-
tional properties and health benefits of bioactive pep-
tides derived from Spirulina: a review, Food Reviews
International 34 (2018) 34e51, https://doi.org/
10.1080/87559129.2016.1210632.
[27] P. Chiaiese, F. Palomba, F. Tatino, C. Lanzillo,
G. Pinto, A. Pollio, E. Filippone, Engineered tobacco
and microalgae secreting the fungal laccase POXA1b
reduce phenol content in olive oil mill wastewater,
Enzyme and Microbial Technology 49 (2011)
540e546, https://doi.org/10.1016/
j.enzmictec.2011.06.002.
[28] S. Godet, C. Loiseau, G. Pencreac’h, F. Ergan,
J. Herault, Isolation and sequence analysis of a cdna
encoding a novel putative esterase from the marine
microalga isochrysis galbana (prymnesiophyceae,
haptophyta), Journal of Phycology 46 (2010)
679e684, https://doi.org/10.1111/j.1529-
8817.2010.00838.x.
[29] S. Godet, J. Herault, G. Pencreac’h, F. Ergan,
C. Loiseau, Isolation and analysis of a gene from the
marine microalga Isochrysis galbana that encodes a
lipase-like protein, Journal of Applied Phycology 24
(2012) 1547e1553, https://doi.org/10.1007/s10811-
012-9815-3.
[30] E. Suarez Garcia, J. van Leeuwen, C. Safi, L. Sijtsma,
M.H.M. Eppink, R.H. Wijffels, C. van den Berg,
IV. Selected examples and case history
Selective and energy efficient extraction of functional
proteins from microalgae for food applications, Bio-
resource Technology 268 (2018) 197e203, https://
doi.org/10.1016/j.biortech.2018.07.131.
[31] E.S. Garcia, J.J.A. Van Leeuwen, C. Safi, L. Sijtsma,
L.A.M. Van Den Broek, M.H.M. Eppink,
R.H. Wijffels, C. Van Den Berg, Techno-Functional
properties of crude extracts from the green microalga
Tetraselmis suecica, Journal of Agricultural and Food
Chemistry 66 (2018) 7831e7838, https://doi.org/
10.1021/acs.jafc.8b01884.
[32] F. Di Caprio, F. Pagnanelli, R.H. Wijffels, D. Van der
Veen, Quantification of Tetradesmus obliquus (Chloro-
phyceae) cell size and lipid content heterogeneity at
single-cell level, Journal of Phycology 54 (2018)
187e197, https://doi.org/10.1111/jpy.12610.
[33] C. Largeau, E. Casadevall, C. Berkaloff,
P. Dhamelincourt, Sites of accumulation and composi-
tion of hydrocarbons in Botryococcus braunii, Phyto-
chemistry 19 (1980) 1043e1051, https://doi.org/
10.1016/0031-9422(80)83054-8.
[34] J. Kim, G. Yoo, H. Lee, J. Lim, K. Kim, C.W. Kim,
M.S. Park, J.W. Yang, Methods of downstream pro-
cessing for the production of biodiesel from
microalgae, Biotechnology Advances 31 (2013)
862e876, https://doi.org/10.1016/
j.biotechadv.2013.04.006.
[35] P. Roesle, F. Stempfle, S.K. Hess, J. Zimmerer,
C. Ríobartulos, B. Lepetit, A. Eckert, P.G. Kroth,
S. Mecking, Synthetic polyester from algae oil, Ange-
wandte Chemie International Edition 53 (2014)
6800e6804, https://doi.org/10.1002/anie.201403991.
[36] B.S. Grama, S.N. Agathos, C.S. Jeffryes, Balancing
photosynthesis and respiration increases microalgal
biomass productivity during photoheterotrophy on
glycerol, ACS Sustainable Chemistry and Engineering
4 (2016) 1611e1618, https://doi.org/10.1021/
acssuschemeng.5b01544.
[37] O.P. Ward, A. Singh, Omega-3/6 fatty acids: alterna-
tive sources of production, Process Biochemistry 40
(2005) 3627e3652, https://doi.org/10.1016/
j.procbio.2005.02.020.
[38] T.C. Adarme-Vega, D.K.Y. Lim, M. Timmins,
F. Vernen, Y. Li, P.M. Schenk, Microalgal biofactories:
a promising approach towards sustainable omega-3
fatty acid production, Microbial Cell Factories 11
(2012), https://doi.org/10.1186/1475-2859-11-96.
[39] R.R. Ambati, P.S. Moi, S. Ravi,
R.G. Aswathanarayana, Astaxanthin: sources, extrac-
tion, stability, biological activities and its commercial
 References
applications e a review, Marine Drugs 12 (2014) [50]
128e152, https://doi.org/10.3390/md12010128.
[40] F. Di Caprio, P. Altimari, L. Toro, F. Pagnanelli, Effect
of lipids and carbohydrates extraction on astaxanthin
stability in Scenedesmus sp, Chemical Engineering
Transactions 43 (2015) 205e210, https://doi.org/ [51]
10.3303/CET1543035.
[41] J.H. Lin, D.J. Lee, J.S. Chang, Lutein production from
biomass: marigold flowers versus microalgae, Bio-
resource Technology 184 (2015) 421e428, https:// [52]
doi.org/10.1016/j.biortech.2014.09.099.
[42] R. Bosma, J.H. de Vree, P.M. Slegers, M. Janssen,
R.H. Wijffels, M.J. Barbosa, Design and construction
of the microalgal pilot facility AlgaePARC, Algal
Research 6 (2014) 160e169, https://doi.org/
10.1016/j.algal.2014.10.006. [53]
[43] F. Bumbak, S. Cook, V. Zachleder, S. Hauser,
K. Kovar, Best practices in heterotrophic high-cell-
density microalgal processes: achievements, potential
and possible limitations, Applied Microbiology and
Biotechnology 91 (2011) 31e46, https://doi.org/ [54]
10.1007/s00253-011-3311-6.
[44] M.L. Gerardo, S. Van Den Hende, H. Vervaeren,
T. Coward, S.C. Skill, Harvesting of microalgae within
a biorefinery approach: a review of the developments [55]
and case studies from pilot-plants, Algal Research 11
(2015) 248e262, https://doi.org/10.1016/
j.algal.2015.06.019.
[45] M. Al Hattab, A. Ghaly, Microalgae oil extraction pre-
treatment methods: critical review and comparative
analysis, Journal of Fundamentals of Renewable En- [56]
ergy and Applications 5 (2015) 1e26, https://
doi.org/10.4172/2090-4541.1000172.
[46] T. Dong, E.P. Knoshaug, P.T. Pienkos, L.M.L. Laurens,
Lipid recovery from wet oleaginous microbial [57]
biomass for biofuel production: a critical review,
Applied Energy 177 (2016) 879e895, https://
doi.org/10.1016/j.apenergy.2016.06.002.
[47] M.A. Hejazi, E. Holwerda, R.H. Wijffels, Milking
microalga Dunaliella salina for b-Carotene production [58]
in two-phase bioreactors, Biotechnology and Bioengi-
neering 85 (2004) 475e481, https://doi.org/10.1002/
bit.10914.
[48] N.R. Moheimani, R. Cord-Ruwisch, E. Raes,
M.A. Borowitzka, Non-destructive oil extraction
from Botryococcus braunii (Chlorophyta), Journal of [59]
Applied Phycology 25 (2013) 1653e1661, https://
doi.org/10.1007/s10811-013-0012-9.
[49] F. Zhang, L.H. Cheng, X.H. Xu, L. Zhang, H.L. Chen,
Screening of biocompatible organic solvents for
enhancement of lipid milking from Nannochloropsis
sp, Process Biochemistry 46 (2011) 1934e1941, [60]
https://doi.org/10.1016/j.procbio.2011.06.024.
IV. Selected examples and case history
309
E. G€unerken, E. D’Hondt, M.H.M. Eppink, L. Garcia-
Gonzalez, K. Elst, R.H. Wijffels, Cell disruption for
microalgae biorefineries, Biotechnology Advances 33
(2015) 243e260, https://doi.org/10.1016/
j.biotechadv.2015.01.008.
R. Halim, M.K. Danquah, P.A. Webley, Extraction of
oil from microalgae for biodiesel production: a
review, Biotechnology Advances 30 (2012) 709e732,
https://doi.org/10.1016/j.biotechadv.2012.01.001.
P.R. Postma, E. Suarez-Garcia, C. Safi, G. Olivieri,
R.H. Wijffels, Energy efficient bead milling of microal-
gae: effect of bead size on disintegration and release of
proteins and carbohydrates, Bioresource Technology
224 (2017) 670e679, https://doi.org/10.1016/
j.biortech.2016.11.071.
M. Wang, W. Yuan, X. Jiang, Y. Jing, Z. Wang, Disrup-
tion of microalgal cells using high-frequency focused
ultrasound, Bioresource Technology 153 (2014)
315e321, https://doi.org/10.1016/
j.biortech.2013.11.054.
F.J. Petracek, L. Zechmeister, Determination of parti-
tion coefficients of carotenoids as a tool in pigment
analysis, Analytical Chemistry 28 (1956) 1484e1485,
https://doi.org/10.1021/ac60117a041.
R. Dineshkumar, S.K. Dash, R. Sen, Process integra-
tion for microalgal lutein and biodiesel production
with concomitant flue gas CO2 sequestration: a bio-
refinery model for healthcare, energy and
environment, RSC Advances 5 (2015) 73381e73394,
https://doi.org/10.1039/c5ra09306f.
M. D’Este, D. De Francisci, I. Angelidaki, Novel proto-
col for lutein extraction from microalga Chlorella
vulgaris, Biochemical Engineering Journal 127 (2017)
175e179, https://doi.org/10.1016/j.bej.2017.06.019.
H. Bin Li, Y. Jiang, F. Chen, Isolation and purification
of lutein from the microalga Chlorella vulgaris by
extraction after saponification, Journal of Agricultural
and Food Chemistry 50 (2002) 1070e1072, https://
doi.org/10.1021/jf010220b.
O.K. Lee, A.L. Kim, D.H. Seong, C.G. Lee, Y.T. Jung,
J.W. Lee, E.Y. Lee, Chemo-enzymatic saccharification
and bioethanol fermentation of lipid-extracted resid-
ual biomass of the microalga, Dunaliella tertiolecta, Bio-
resource Technology 132 (2013) 197e201, https://
doi.org/10.1016/j.biortech.2013.01.007.
D. Hernandez, M. Solana, B. Ria~ no, M.C. García-
Gonzalez, A. Bertucco, Biofuels from microalgae: lipid
extraction and methane production from the residual
biomass in a biorefinery approach, Bioresource Tech-
nology 170 (2014) 370e378, https://doi.org/10.1016/
j.biortech.2014.07.109.
M. Francavilla, P. Kamaterou, S. Intini,
M. Monteleone, A. Zabaniotou, Cascading microalgae
310 16. New strategies enhancing feasibility of microalgal cultivations
biorefinery: fast pyrolysis of Dunaliella tertiolecta
lipid extracted-residue, Algal Research 11 (2015)
184e193, https://doi.org/10.1016/
j.algal.2015.06.017.
[61] Y. Zhu, K.O. Albrecht, D.C. Elliott, R.T. Hallen,
S.B. Jones, Development of hydrothermal liquefaction
and upgrading technologies for lipid-extracted algae
conversion to liquid fuels, Algal Research 2 (2013)
455e464, https://doi.org/10.1016/
j.algal.2013.07.003.
[62] E.J. Kim, S. Park, H.J. Hong, Y.E. Choi, J.W. Yang, Bio-
sorption of chromium (Cr(III)/Cr(VI)) on the residual
microalga Nannochloris oculata after lipid extraction
for biodiesel production, Bioresource Technology
102 (2011) 11155e11160, https://doi.org/10.1016/
j.biortech.2011.09.107.
[63] J.A. Gerde, T. Wang, L. Yao, S. Jung, L.A. Johnson,
B. Lamsal, Optimizing protein isolation from defatted
and non-defatted Nannochloropsis microalgae
biomass, Algal Research 2 (2013) 145e153, https://
doi.org/10.1016/j.algal.2013.02.001.
[64] A. Visca, F. Di Caprio, R. Spinelli, P. Altimari, A. Cicci,
G. Iaquaniello, L. Toro, F. Pagnanelli, Microalgae
cultivation for lipids and carbohydrates production,
Chemical Engineering Transactions 57 (2017)
127e132, https://doi.org/10.3303/CET1757022.
[65] N. Lumdubwong, P.A. Seib, Rice starch isolation by
alkaline protease digestion of wet-milled rice flour,
Journal of Cereal Science 31 (2000) 63e74, https://
doi.org/10.1006/jcrs.1999.0279.
[66] S. Lim, J.-H. Lee, D.-H. Shin, H.S. Lim, Comparison of
protein extraction solutions for rice starch isolation
and effects of residual protein content on starch
pasting properties, Starch Staerke 51 (1999) 120e125,
https://doi.org/10.1002/(SICI)1521-379X(199904)51:
4<120::AID-STAR120>3.3.CO;2-1.
[67] R.K. Desai, H. Monteillet, X. Li, B. Schuur, J.M. Kleijn,
F.A.M. Leermakers, R.H. Wijffels, M.H.M. Eppink,
One-step mild biorefinery of functional biomolecules
from microalgae extracts, Reaction Chemistry and En-
gineering 3 (2018) 182e187, https://doi.org/10.1039/
c7re00116a.
[68] L. Soto-Sierra, P. Stoykova, Z.L. Nikolov, Extraction
and fractionation of microalgae-based protein
products, Algal Research 36 (2018) 175e192,
https://doi.org/10.1016/j.algal.2018.10.023.
[69] L. Grossmann, S. Ebert, J. Hinrichs, J. Weiss, Effect of
precipitation, lyophilization, and organic solvent
extraction on preparation of protein-rich powders
from the microalgae Chlorella protothecoides, Algal
Research 29 (2018) 266e276, https://doi.org/
10.1016/j.algal.2017.11.019.
IV. Selected examples and case history
[70] W. Blanken, P.R. Postma, L. de Winter, R.H. Wijffels,
M. Janssen, Predicting microalgae growth, Algal
Research 14 (2016) 28e38, https://doi.org/10.1016/
j.algal.2015.12.020.
[71] F. Pagnanelli, P. Altimari, F. Trabucco, L. Toro, Mixo-
trophic growth of Chlorella vulgaris and Nannochlorop-
sis oculata: interaction between glucose and nitrate,
Journal of Chemical Technology and Biotechnology
89 (2014) 652e661, https://doi.org/10.1002/
jctb.4179.
[72] A. Mazzelli, A. Cicci, G. Franceschini, F.D. Caprio,
G. Iaquaniello, P. Altimari, F. Pagnanelli, L. Toro,
Investigation of effects of nutrients and external pa-
rameters on kinetic growth of outdoor microalgal
cultivation, Chemical Engineering Transactions 64
(2018), https://doi.org/10.3303/CET1864116.
[73] S. Egloff, F. Tschudi, Z. Schmautz, D. Refardt, High-
density cultivation of microalgae continuously fed
with unfiltered water from a recirculating aquaculture
system, Algal Research 34 (2018) 68e74, https://
doi.org/10.1016/j.algal.2018.07.004.
[74] J. Doucha, K. Lívanský, High density outdoor micro-
algal culture, in: Algal Biorefineries, vol. 1, Cultiv.
Cells Prod., 2014, pp. 147e173, https://doi.org/
10.1007/978-94-007-7494-0_6.
[75] A.C. Apel, C.E. Pfaffinger, N. Basedahl,
N. Mittwollen, J. G€ obel, J. Sauter, T. Br€ uck,
D. Weuster-Botz, Open thin-layer cascade reactors
for saline microalgae production evaluated in a phys-
ically simulated Mediterranean summer climate,
Algal Research 25 (2017) 381e390, https://doi.org/
10.1016/j.algal.2017.06.004.
[76] I. Gifuni, A. Pollio, A. Marzocchella, G. Olivieri, New
ultra-flat photobioreactor for intensive microalgal
production: the effect of light irradiance, Algal
Research 34 (2018) 134e142, https://doi.org/
10.1016/j.algal.2018.07.014.
[77] C.J. Hulatt, D.N. Thomas, Energy efficiency of an out-
door microalgal photobioreactor sited at mid-
temperate latitude, Bioresource Technology 102
(2011) 6687e6695, https://doi.org/10.1016/
j.biortech.2011.03.098.
[78] T. Naumann, Z. Çebi, B. Podola, M. Melkonian,
Growing microalgae as aquaculture feeds on twin-
layers: a novel solid-state photobioreactor, Journal of
Applied Phycology 25 (2013) 1413e1420, https://
doi.org/10.1007/s10811-012-9962-6.
[79] L.B. Christenson, R.C. Sims, Rotating algal biofilm
reactor and spool harvester for wastewater treatment
with biofuels by-products, Biotechnology and Bioen-
gineering 109 (2012) 1674e1684, https://doi.org/
10.2478/mspe-2018-0005.
 References
[80] W. Blanken, M. Janssen, M. Cuaresma, Z. Libor, [90]
T. Bhaiji, R.H. Wijffels, Biofilm growth of Chlorella soro-
kiniana in a rotating biological contactor based
photobioreactor, Biotechnology and Bioengineering
111 (2014) 2436e2445, https://doi.org/10.1002/
bit.25301.
[81] P.J. Graham, B. Nguyen, S.C. Pierobon, X. Cheng, [91]
E.G. Karakolis, D. Sinton, Emerging microalgae tech-
nology: a review, Sustainable Energy and Fuels
(2017), https://doi.org/10.1039/C7SE00236J.
[82] B. Podola, T. Li, M. Melkonian, Porous substrate bio-
reactors: a paradigm shift in microalgal
biotechnology? Trends in Biotechnology 35 (2017) [92]
121e132, https://doi.org/10.1016/
j.tibtech.2016.06.004.
[83] T. Li, G. Lin, B. Podola, M. Melkonian, Continuous
removal of zinc from wastewater and mine dump
leachate by a microalgal biofilm PSBR, Journal of Haz-
ardous Materials 297 (2015) 112e118, https://
doi.org/10.1016/j.jhazmat.2015.04.080. [93]
[84] L. Zhang, L. Chen, J. Wang, Y. Chen, X. Gao,
Z. Zhang, T. Liu, Attached cultivation for improving
the biomass productivity of Spirulina platensis, Bio-
resource Technology 181 (2015) 136e142, https://
doi.org/10.1016/j.biortech.2015.01.025.
[85] T. Liu, J. Wang, Q. Hu, P. Cheng, B. Ji, J. Liu, Y. Chen, [94]
W. Zhang, X. Chen, L. Chen, L. Gao, C. Ji, H. Wang,
Attached cultivation technology of microalgae for effi-
cient biomass feedstock production, Bioresource
Technology 127 (2013) 216e222, https://doi.org/
10.1016/j.biortech.2012.09.100. [95]
[86] L.K.P. Schultze, M.V. Simon, T. Li, D. Langenbach,
B. Podola, M. Melkonian, High light and carbon diox-
ide optimize surface productivity in a twin-layer bio-
film photobioreactor, Algal Research 8 (2015) 37e44,
https://doi.org/10.1016/j.algal.2015.01.007.
[87] A. Janoska, P.P. Lamers, A. Hamhuis, Y. van Eimeren, [96]
R.H. Wijffels, M. Janssen, A liquid foam-bed photo-
bioreactor for microalgae production, Chemical Engi-
neering Journal 313 (2017) 1206e1214, https://
doi.org/10.1016/j.cej.2016.11.022.
[88] A. Janoska, M. Vazquez, M. Janssen, R.H. Wijffels,
M. Cuaresma, C. Vílchez, Surfactant selection for a
liquid foam-bed photobioreactor, Biotechnology [97]
Progress 34 (2018) 711e720, https://doi.org/
10.1002/btpr.2614.
[89] A. Janoska, R. Barten, S. de Nooy, P. van Rijssel,
R.H. Wijffels, M. Janssen, Improved liquid foam-bed
photobioreactor design for microalgae cultivation,
Algal Research 33 (2018) 55e70, https://doi.org/
10.1016/j.algal.2018.04.025.
IV. Selected examples and case history
311
E.G. Nwoba, D.A. Parlevliet, D.W. Laird, K. Alameh,
N.R. Moheimani, Sustainable phycocyanin produc-
tion from Arthrospira platensis using solar-control
thin film coated photobioreactor, Biochemical Engi-
neering Journal 141 (2019) 232e238, https://
doi.org/10.1016/j.bej.2018.10.024.
E.E. Jung, A. Jain, N. Voulis, D.F.R. Doud,
L.T. Angenent, D. Erickson, Stacked optical wave-
guide photobioreactor for high density algal
cultures, Bioresource Technology 171 (2014)
495e499, https://doi.org/10.1016/
j.biortech.2014.08.093.
Y. Sun, Q. Liao, Y. Huang, A. Xia, Q. Fu, X. Zhu,
Y. Zheng, Integrating planar waveguides doped
with light scattering nanoparticles into a flat-plate
photobioreactor to improve light distribution and
microalgae growth, Bioresource Technology 220
(2016) 215e224, https://doi.org/10.1016/
j.biortech.2016.08.063.
S. Karuppasamy, A.S. Musale, B. Soni, B. Bhadra,
N. Gujarathi, M. Sundaram, A. Sapre, S. Dasgupta,
C. Kumar, Integrated grazer management mediated
by chemicals for sustainable cultivation of algae in
open ponds, Algal Research 35 (2018) 439e448,
https://doi.org/10.1016/j.algal.2018.09.017.
R. Singh, R. Birru, G. Sibi, Nutrient removal effi-
ciencies of Chlorella vulgaris from the urban waste-
water for reduced eutrofication, Journal of
Environmental Protection 8 (2017) 1e11, https://
doi.org/10.4236/jep.2017.81001.
M.P. Caporgno, A. Taleb, M. Olkiewicz, J. Font,
J. Pruvost, J. Legrand, C. Bengoa, Microalgae cultiva-
tion in urban wastewater: nutrient removal and
biomass production for biodiesel and methane, Algal
Research 10 (2015) 232e239, https://doi.org/
10.1016/j.algal.2015.05.011.
K. Katam, D. Bhattacharyya, Comparative study on
treatment of kitchen wastewater using a mixed
microalgal culture and an aerobic bacterial culture:
kinetic evaluation and FAME analysis, Environ-
mental Science and Pollution Research 25 (2018)
20732e20742, https://doi.org/10.1007/s11356-018-
2209-6.
C. Gonzalez-Fernandez, B. Ria~
no-Irazabal,
B. Molinuevo-Salces, S. Blanco, M.C. García-
Gonzalez, Effect of operational conditions on the
degradation of organic matter and development of
microalgae-bacteria consortia when treating swine
slurry, Applied Microbiology and Biotechnology 90
(2011) 1147e1153, https://doi.org/10.1007/s00253-
011-3111-z.
312 16. New strategies enhancing feasibility of microalgal cultivations
[98] X. Han, N. Rusconi, P. Ali, K. Pagkatipunan, F. Chen,
Nutrients extracted from chicken manure accelerate
growth of microalga Scenedesmus obliquus HTB1,
Green and Sustainable Chemistry 7 (2017) 101e113,
https://doi.org/10.4236/gsc.2017.72009.
[99] M. Franchino, E. Comino, F. Bona, V.A. Riggio, Che-
mosphere growth of three microalgae strains and
nutrient removal from an agro-zootechnical
digestate, Chemosphere 92 (2013) 738e744, https://
doi.org/10.1016/j.chemosphere.2013.04.023.
[100] A. Ruiz-Martinez, N. Martin Garcia, I. Romero,
A. Seco, J. Ferrer, Microalgae cultivation in waste-
water: nutrient removal from anaerobic membrane
bioreactor effluent, Bioresource Technology 126
(2012) 247e253, https://doi.org/10.1016/j.biortech.
2012.09.022.
[101] A.S. Vikneswara, R.M.S. Radin Mohamed, A.A.S. Al-
Gheethi, A.H. Mohd Kassim, N. Othman, Removal of
Nutrients from Meat Processing Wastewater Through
the Phycoremediation Process. Management of Grey-
water in Developing Countries, Springer International
Publishing, 2019, pp. 245e263, https://doi.org/
10.1007/978-3-319-90269-2_13.
[102] J.M. Girard, R. Tremblay, N. Faucheux, M. Heitz,
J.S. Desch^enes, Phycoremediation of cheese whey
permeate using directed commensalism between Sce-
nedesmus obliquus and Chlorella protothecoides, Algal
Research 22 (2017) 122e126, https://doi.org/
10.1016/j.algal.2016.12.013.
[103] J.M. Girard, M.L. Roy, M. Ben Hafsa, J. Gagnon,
N. Faucheux, M. Heitz, R. Tremblay, J.S. Desch^enes,
Mixotrophic cultivation of green microalgae Scenedes-
mus obliquus on cheese whey permeate for biodiesel
production, Algal Research 5 (2014) 241e248,
https://doi.org/10.1016/j.algal.2014.03.002.
[104] F. Di Caprio, P. Altimari, G. Iaquaniello, L. Toro,
F. Pagnanelli, T. Obliquus mixotrophic cultivation in
treated and untreated olive mill wastewater, Chemi-
cal Engineering Transactions 64 (2018), https://
doi.org/10.3303/CET1864105.
[105] F. Di Caprio, P. Altimari, F. Pagnanelli, Integrated
microalgae biomass production and olive mill waste-
water biodegradation: optimization of the wastewater
supply strategy, Chemical Engineering Journal 349
(2018) 539e546, https://doi.org/10.1016/
j.cej.2018.05.084.
[106] C.J. de Andrade, L.M. de Andrade, Microalgae for
bioremediation of textile wastewater: an overview,
MOJ Food Processing and Technology 6 (2018),
https://doi.org/10.15406/mojfpt.2018.06.00200.
[107] L. Xin, H. Hong-ying, G. Ke, S. Ying-xue, Effects of
different nitrogen and phosphorus concentrations
on the growth, nutrient uptake, and lipid
IV. Selected examples and case history
accumulation of a freshwater microalga Scenedes-
mus sp, Bioresource Technology 101 (2010)
5494e5500, https://doi.org/10.1016/j.biortech.
2010.02.016.
[108] R.W. Krauss, W.H. Thomas, The growth and inor-
ganic nutrition of Scenedesmus obliquus in mass cul-
ture. 123, Plant Physiology 29 (1954) 205e214,
https://doi.org/10.1104/pp.29.3.205.
[109] R.W. Sterner, J.J. Elser, Stoichiometry and
homeostasis, Ecological Stoichiometry (2002) 1e43,
https://doi.org/10.1073/pnas.0703993104.
[110] F. Di Caprio, P. Scarponi, P. Altimari, G. Iaquaniello,
F. Pagnanelli, The influence of phenols extracted
from olive mill wastewater on the heterotrophic
and mixotrophic growth of Scenedesmus sp, Journal
of Chemical Technology and Biotechnology 93
(2018) 3619e3626, https://doi.org/10.1002/
jctb.5743.
[111] D.L. Cheng, H.H. Ngo, W.S. Guo, S.W. Chang,
D.D. Nguyen, S.M. Kumar, Microalgae biomass from
swine wastewater and its conversion to bioenergy, Bio-
resource Technology 275 (2019) 109e122, https://
doi.org/10.1016/j.biortech.2018.12.019.
[112] T. Cai, S.Y. Park, Y. Li, Nutrient recovery from waste-
water streams by microalgae: status and prospects,
Renewable and Sustainable Energy Reviews 19
(2013) 360e369, https://doi.org/10.1016/
j.rser.2012.11.030.
[113] O. Bernard, Hurdles and challenges for modelling and
control of microalgae for CO2 mitigation and biofuel
production, Journal of Process Control 21 (2011)
1378e1389, https://doi.org/10.1016/j.jprocont.
2011.07.012.
[114] F. Di Caprio, P. Altimari, F. Pagnanelli, Effect of Ca2þ
concentration on Scenedesmus sp. growth in heterotro-
phic and photoautotrophic cultivation, Nature
Biotechnology 40 B (2017) 228e235, https://
doi.org/10.1016/j.nbt.2017.09.003.
[115] T. K€allqvist, A. Svenson, Assessment of ammonia
toxicity in tests with the microalga, Nephroselmis pyrifor-
mis, Chlorophyta, Water Research 37 (2003) 477e484,
https://doi.org/10.1016/S0043-1354(02)00361-5.
[116] A. Xia, J.D. Murphy, Microalgal cultivation in treating
liquid digestate from biogas systems, Trends in
Biotechnology 34 (2016) 264e275, https://doi.org/
10.1016/j.tibtech.2015.12.010.
[117] G. Hodaifa, M.E. Martínez, S. Sanchez, Influence of
temperature on growth of Scenedesmus obliquus in
diluted olive mill wastewater as culture medium, En-
gineering in Life Science 10 (2010) 257e264, https://
doi.org/10.1002/elsc.201000005.
[118] F. Di Caprio, P. Altimari, F. Pagnanelli, Integrated
biomass production and biodegradation of olive mill
 References
wastewater by cultivation of Scenedesmus sp, Algal
Research 9 (2015) 306e311.
[119] G. Markou, I. Chatzipavlidis, D. Georgakakis, Culti-
vation of Arthrospira (Spirulina) platensis in olive-
oil mill wastewater treated with sodium
hypochlorite, Bioresource Technology 112 (2012)
234e241, https://doi.org/10.1016/
j.biortech.2012.02.098.
[120] G. Hodaifa, S. Sanchez, M.E. Martínez, R. Orpez,
Biomass production of Scenedesmus obliquus from mix-
tures of urban and olive-oil mill wastewaters used as
culture medium, Applied Energy 104 (2013) 345e352,
https://doi.org/10.1016/j.apenergy.2012.11.005.
[121] B. Farizoglu, B. Keskinler, E. Yildiz, A. Nuhoglu,
Cheese whey treatment performance of an aerobic
jet loop membrane bioreactor, Process Biochemistry
39 (2004) 2283e2291, https://doi.org/10.1016/
j.procbio.2003.11.028.
[122] Y. Zhang, A. Kendall, J. Yuan, A comparison of on-site
nutrient and energy recycling technologies in algal oil
production, Resources, Conservation and Recycling
88 (2014) 13e20, https://doi.org/10.1016/
j.resconrec.2014.04.011.
[123] F. Di Caprio, P. Altimari, D. Uccelletti, F. Pagnanelli,
Mechanistic modelling of copper biosorption by
wild type and engineered Saccharomyces cerevisiae
biomasses, Chemical Engineering Journal 244 (2014)
561e568.
[124] G. Markou, L. Wang, J. Ye, A. Unc, Using agro-
industrial wastes for the cultivation of microalgae
and duckweeds: contamination risks and biomass
safety concerns, Biotechnology Advances 36 (2018)
1238e1254, https://doi.org/10.1016/
j.biotechadv.2018.04.003.
[125] F. Di Caprio, P. Altimari, E. Zanni, D. Uccelletti,
L. Toro, F. Pagnanelli, Lanthanum biosorption by
different saccharomyces cerevisiae strains, Chemical
Engineering Transactions 49 (2016) 37e42, https://
doi.org/10.3303/CET1649007.
[126] F.Z. Mennaa, Z. Arbib, J.A. Perales, Urban waste-
water treatment by seven species of microalgae and
analgal bloom: biomass production, N and P
removal kinetics and harvestability, Water Research
83 (2015) 42e51, https://doi.org/10.1016/
j.watres.2015.06.007.
[127] S.Y. Chiu, C.Y. Kao, T.Y. Chen, Y. Bin Chang,
C.M. Kuo, C.S. Lin, Cultivation of microalgal Chlor-
ella for biomass and lipid production using waste-
water as nutrient resource, Bioresource Technology
184 (2015) 179e189, https://doi.org/10.1016/
j.biortech.2014.11.080.
IV. Selected examples and case history
313
[128] J.C.M. Pires, M.C.M. Alvim-Ferraz, F.G. Martins,
M. Sim~ oes, Wastewater treatment to enhance the eco-
nomic viability of microalgae culture, Environmental
Science and Pollution Research 20 (2013) 5096e5105,
https://doi.org/10.1007/s11356-013-1791-x.
[129] P. Foladori, S. Petrini, G. Andreottola, Evolution of
real municipal wastewater treatment in photobioreac-
tors and microalgae-bacteria consortia using real-time
parameters, Chemical Engineering Journal 345 (2018)
507e516, https://doi.org/10.1016/j.cej.2018.03.178.
[130] A.L. Gonçalves, J.C.M. Pires, M. Sim~oes, A review on
the use of microalgal consortia for wastewater
treatment, Algal Research 24 (2017) 403e415,
https://doi.org/10.1016/j.algal.2016.11.008.
[131] M.H. Huang, Y.M. Li, G.W. Gu, Chemical composi-
tion of organic matters in domestic wastewater, Desa-
lination 262 (2010) 36e42, https://doi.org/10.1016/
j.desal.2010.05.037.
[132] L. Peng, H.H. Ngo, W.S. Guo, Y. Liu, D. Wang,
S. Song, W. Wei, L.D. Nghiem, B.J. Ni, A novel mech-
anistic model for nitrogen removal in algal-bacterial
photo sequencing batch reactors, Bioresource Tech-
nology 267 (2018) 502e509, https://doi.org/
10.1016/j.biortech.2018.07.093.
[133] P. Foladori, S. Petrini, M. Nessenzia, G. Andreottola,
Enhanced nitrogen removal and energy saving in a
microalgal-bacterial consortium treating real munic-
ipal wastewater, Water Science and Technology 78
(2018) 174e182, https://doi.org/10.2166/
wst.2018.094.
[134] J. Ye, J. Liang, L. Wang, G. Markou, Q. Jia, Operation
optimization of a photo-sequencing batch reactor for
wastewater treatment: study on influencing factors
and impact on symbiotic microbial ecology, Bio-
resource Technology 252 (2018) 7e13, https://
doi.org/10.1016/j.biortech.2017.12.086.
[135] M.A. Alam, C. Wan, X.Q. Zhao, L.J. Chen, J.S. Chang,
F.W. Bai, Enhanced removal of Zn2þ or Cd2þ by the
flocculating Chlorella vulgaris JSC-7, Journal of Haz-
ardous Materials 289 (2015) 38e45, https://doi.org/
10.1016/j.jhazmat.2015.02.012.
[136] O. Perez-Garcia, F.M.E. Escalante, L.E. de-Bashan,
Y. Bashan, Heterotrophic cultures of microalgae:
metabolism and potential products, Water Research
45 (2011) 11e36, https://doi.org/10.1016/
j.watres.2010.08.037.
[137] S. Wang, Y. Wu, X. Wang, Heterotrophic cultivation
of Chlorella pyrenoidosa using sucrose as the sole car-
bon source by co-culture with Rhodotorula glutinis,
Bioresource Technology 220 (2016) 615e620,
https://doi.org/10.1016/j.biortech.2016.09.010.
314 16. New strategies enhancing feasibility of microalgal cultivations
[138] S. Ethier, K. Woisard, D. Vaughan, Z. Wen, Contin-
uous culture of the microalgae Schizochytrium limaci-
num on biodiesel-derived crude glycerol for
producing docosahexaenoic acid, Bioresource Tech-
nology 102 (2011) 88e93, https://doi.org/10.1016/
j.biortech.2010.05.021.
[139] C. Baroukh, V. Turon, O. Bernard, Dynamic metabolic
modeling of heterotrophic and mixotrophic microal-
gal growth on fermentative wastes, PLoS Computa-
tional Biology (2017) 1e18, https://doi.org/
10.1371/journal.pcbi.1005590.
[140] J.S. Desch^enes, A bacteriostatic control approach for
mixotrophic cultures of microalgae,
IFAC-PapersOnLine 49 (2016) 1074e1078, https://
doi.org/10.1016/j.ifacol.2016.07.345.
[141] P. Bohutskyi, K. Liu, L.K. Nasr, N. Byers,
J.N. Rosenberg, G.A. Oyler, M.J. Betenbaugh,
E.J. Bouwer, Bioprospecting of microalgae for inte-
grated biomass production and phytoremediation of
unsterilized wastewater and anaerobic digestion
centrate, Applied Microbiology and Biotechnology
99 (2015) 6139e6154, https://doi.org/10.1007/
s00253-015-6603-4.
[142] G. Mujtaba, K. Lee, Treatment of real wastewater us-
ing co-culture of immobilized Chlorella vulgaris and
suspended activated sludge, Water Research 120
(2017) 174e184, https://doi.org/10.1016/
j.watres.2017.04.078.
[143] M.R. Droop, 25 Years of algal growth kinetics,
Botanica Marina 26 (1983) 99e112, https://doi.org/
10.1515/botm.1983.26.3.99.
[144] J.S. Desch^enes, A. Boudreau, R. Tremblay, Mixotro-
phic production of microalgae in pilot-scale photo-
bioreactors: practicability and process
considerations, Algal Research 10 (2015) 80e86,
https://doi.org/10.1016/j.algal.2015.04.015.
[145] G.S. Khush, Green revolution: the way forward, Na-
ture Reviews Genetics 2 (2001) 815e822, https://
doi.org/10.1038/35093585.
[146] H.H. Iltis, From teosinte to maize: the catastrophic
sexual transmutation, Science 222 (1983) 886e894,
https://doi.org/10.1126/science.222.4626.886.
[147] P.G. Stephenson, C.M. Moore, M.J. Terry,
M.V. Zubkov, T.S. Bibby, Improving photosynthesis
for algal biofuels: toward a green revolution, Trends
in Biotechnology 29 (2011) 615e623, https://
doi.org/10.1016/j.tibtech.2011.06.005.
[148] J.E.W. Polle, S. Kanakagiri, E.S. Jin, T. Masuda,
A. Melis, Truncated chlorophyll antenna size of the
photosystems e a practical method to improve micro-
algal productivity and hydrogen production in mass
culture, in: Int. J. Hydrogen Energy, 2002,
IV. Selected examples and case history
pp. 1257e1264, https://doi.org/10.1016/S0360-
3199(02)00116-7.
[149] P. Altimari, F. Di Caprio, L. Toro, A.L. Capriotti,
F. Pagnanelli, Hydrogen photo-production by mixo-
trophic cultivation of Chlamydomonas reinhardtii: inter-
action between organic carbon and nitrogen,
Chemical Engineering Transactions 38 (2014)
199e204, https://doi.org/10.3303/CET1438034.
[150] S.N. Kosourov, M.L. Ghirardi, M. Seibert, A truncated
antenna mutant of Chlamydomonas reinhardtii can pro-
duce more hydrogen than the parental strain, Interna-
tional Journal of Hydrogen Energy 36 (2011)
2044e2048, https://doi.org/10.1016/
j.ijhydene.2010.10.041.
[151] A. Melis, Solar energy conversion efficiencies in
photosynthesis: minimizing the chlorophyll antennae
to maximize efficiency, Plant Science 177 (2009)
272e280, https://doi.org/10.1016/
j.plantsci.2009.06.005.
[152] R. Radakovits, R.E. Jinkerson, A. Darzins,
M.C. Posewitz, Genetic engineering of algae for
enhanced biofuel production, Eukaryotic Cell 9 (2010)
486e501, https://doi.org/10.1128/EC.00364-09.
[153] G. Breuer, L. de Jaeger, R.E. Verbeek, R.B. Draaisma,
D.E. Martens, J. Springer, G. Eggink, R.H. Wijffels, Su-
perior triacylglycerol (TAG) accumulation in starch-
less mutants of Scenedesmus obliquus: (I) mutant
generation and characterization, Biotechnology for
Biofuels 7 (2014) 69, https://doi.org/10.1186/1754-
6834-7-69.
[154] Y.H. Ma, X. Wang, Y.F. Niu, Z.K. Yang, M.H. Zhang,
Z.M. Wang, W.D. Yang, J.S. Liu, H.Y. Li, Antisense
knockdown of pyruvate dehydrogenase kinase pro-
motes the neutral lipid accumulation in the diatom
Phaeodactylum tricornutum, Microbial Cell Factories 13
(2014), https://doi.org/10.1186/s12934-014-0100-9.
[155] J. Xue, Y.F. Niu, T. Huang, W.D. Yang, J.S. Liu,
H.Y. Li, Genetic improvement of the microalga Phaeo-
dactylum tricornutum for boosting neutral lipid
accumulation, Metabolic Engineering 27 (2015) 1e9,
https://doi.org/10.1016/j.ymben.2014.10.002.
[156] K.M. Koo, S. Jung, B.S. Lee, J.B. Kim, Y.D. Jo, H. Il
Choi, S.Y. Kang, G.H. Chung, W.J. Jeong, J.W. Ahn,
The mechanism of starch over-accumulation in Chla-
mydomonas reinhardtii high-starch mutants identified
by comparative transcriptome analysis, Frontiers in
Microbiology 8 (2017), https://doi.org/10.3389/
fmicb.2017.00858.
[157] M. Schulz-Raffelt, V. Chochois, P. Auroy, S. Cuine,
E. Billon, D. Dauvillee, Y. Li-Beisson, G. Peltier, Hy-
per-accumulation of starch and oil in a Chlamydomonas
mutant affected in a plant-specific DYRK kinase,
 References
Biotechnology for Biofuels 9 (2016), https://doi.org/
10.1186/s13068-016-0469-2.
[158] B.F. Cordero, I. Couso, R. Le on, H. Rodríguez, [166]
M.A. Vargas, Enhancement of carotenoids biosyn-
thesis in Chlamydomonas reinhardtii by nuclear trans-
formation using a phytoene synthase gene isolated
from Chlorella zofingiensis, Applied Microbiology and
Biotechnology 91 (2011) 341e351, https://doi.org/
10.1007/s00253-011-3262-y. [167]
[159] P. Perez-L
opez, J.H. de Vree, G. Feijoo, R. Bosma,
M.J. Barbosa, M.T. Moreira, R.H. Wijffels, A.J.B. van
Boxtel, D.M.M. Kleinegris, Comparative life cycle
assessment of real pilot reactors for microalgae culti-
vation in different seasons, Applied Energy 205
(2017) 1151e1164, https://doi.org/10.1016/ [168]
j.apenergy.2017.08.102.
[160] T.M. Lee, Y.F. Tseng, C.L. Cheng, Y.C. Chen, C.S. Lin,
H.Y. Su, T.J. Chow, C.Y. Chen, J.S. Chang, Character-
ization of a heat-tolerant Chlorella sp. GD mutant
with enhanced photosynthetic CO2 fixation efficiency
and its implication as lactic acid fermentation [169]
feedstock, Biotechnology for Biofuels 10 (2017),
https://doi.org/10.1186/s13068-017-0905-y.
[161] P. Mehta, R. Rani, R. Gupta, A.S. Mathur, S.K. Puri,
Biomass and lipid production of a novel freshwater
thermo-tolerant mutant strain of Chlorella pyrenoidosa [170]
NCIM 2738 in seawater salinity recycled medium,
Algal Research 36 (2018) 88e95, https://doi.org/
10.1016/j.algal.2018.10.015.
[162] J. Guan, S. Shen, H. Wu, X. Liu, W. Shen, Y. He,
R. Duan, Biomass and terpenoids produced by [171]
mutant strains of Arthrospira under low temperature
and light conditions, World Journal of Microbiology
and Biotechnology 33 (2017), https://doi.org/
10.1007/s11274-016-2199-9.
[163] M. Kwak, W.K. Park, S.E. Shin, H.G. Koh, B. Lee,
B. ryool Jeong, Y.K. Chang, Improvement of biomass [172]
and lipid yield under stress conditions by using
diploid strains of Chlamydomonas reinhardtii, Algal
Research 26 (2017) 180e189, https://doi.org/
10.1016/j.algal.2017.07.027.
[164] S.O. Kotchoni, E.W. Gachomo, K. Slobodenko,
D.H. Shain, AMP deaminase suppression increases [173]
biomass, cold tolerance and oil content in green
algae, Algal Research 16 (2016) 473e480, https://
doi.org/10.1016/j.algal.2016.04.007.
[165] H.Y. Su, H.H. Chou, T.J. Chow, T.M. Lee, J.S. Chang,
W.L. Huang, H.J. Chen, Improvement of outdoor cul-
ture efficiency of cyanobacteria by over-expression of [174]
stress tolerance genes and its implication as bio-
refinery feedstock, Bioresource Technology 244
IV. Selected examples and case history
315
(2017) 1294e1303, https://doi.org/10.1016/
j.biortech.2017.04.074.
D. Tang, W. Han, P. Li, X. Miao, J. Zhong, CO2 bio-
fixation and fatty acid composition of Scenedesmus
obliquus and Chlorella pyrenoidosa in response to
different CO2 levels, Bioresource Technology 102
(2011) 3071e3076, https://doi.org/10.1016/
j.biortech.2010.10.047.
D. Li, L. Wang, Q. Zhao, W. Wei, Y. Sun, Improving
high carbon dioxide tolerance and carbon dioxide fix-
ation capability of Chlorella sp. by adaptive laboratory
evolution, Bioresource Technology 185 (2015)
269e275, https://doi.org/10.1016/
j.biortech.2015.03.011.
P. Varshney, S. Sohoni, P.P. Wangikar, J. Beardall, Ef-
fect of high CO2 concentrations on the growth and
macromolecular composition of a heat- and high-
light-tolerant microalga, Journal of Applied
Phycology 28 (2016) 2631e2640, https://doi.org/
10.1007/s10811-016-0797-4.
S. Arata, C. Strazza, A. Lodi, A. Del Borghi, Spirulina
platensis culture with flue gas feeding as a
cyanobacteria-based carbon sequestration option,
Chemical Engineering and Technology 36 (2013)
91e97, https://doi.org/10.1002/ceat.201100722.
D. Cheng, X. Li, Y. Yuan, C. Yang, T. Tang, Q. Zhao,
Y. Sun, Adaptive evolution and carbon dioxide fixa-
tion of Chlorella sp. in simulated flue gas, The Science
of the Total Environment 650 (2019) 2931e2938,
https://doi.org/10.1016/j.scitotenv.2018.10.070.
L. Zhou, Y. Yuan, X. Li, S. Mei, J. Gao, Q. Zhao,
W. Wei, Y. Sun, Exploration of phenol tolerance mech-
anism through antioxidative responses of an evolved
strain, Chlorella sp. L5, Journal of Applied Phycology
30 (2018) 2379e2385, https://doi.org/10.1007/
s10811-018-1428-z.
A.A. Corcoran, M.A. Saunders, A.P. Hanley, P.A. Lee,
S. Lopez, R. Ryan, C.B. Yohn, Iterative screening of an
evolutionary engineered Desmodesmus generates
robust field strains with pesticide tolerance, Algal
Research 31 (2018) 443e453, https://doi.org/
10.1016/j.algal.2018.02.026.
A. Ibuot, A.P. Dean, O.A. McIntosh, J.K. Pittman,
Metal bioremediation by CrMTP4 over-expressing
Chlamydomonas reinhardtii in comparison to natural
wastewater-tolerant microalgae strains, Algal
Research 24 (2017) 89e96, https://doi.org/10.1016/
j.algal.2017.03.002.
M.C. Ruiz-Domínguez, I. Vaquero, V. Obreg on, B. de
la Morena, C. Vílchez, J.M. Vega, Lipid accumulation
and antioxidant activity in the eukaryotic acidophilic
316 16. New strategies enhancing feasibility of microalgal cultivations

microalga Coccomyxa sp. (strain onubensis) under
nutrient starvation, Journal of Applied Phycology 27
(2015) 1099e1108, https://doi.org/10.1007/s10811-
014-0403-6.
[175] V. L
opez-Rodas, F. Marva, E. Costas, A. Flores-Moya,
Microalgal adaptation to a stressful environment
(acidic, metal-rich mine waters) could be due to selec-
tion of pre-selective mutants originating in
non-extreme environments, Environmental and
Experimental Botany 64 (2008) 43e48, https://
doi.org/10.1016/j.envexpbot.2008.01.001.
[176] N. von Alvensleben, M. Magnusson, K. Heimann,
Salinity tolerance of four freshwater microalgal spe-
cies and the effects of salinity and nutrient limitation
on biochemical profiles, Journal of Applied Phycology
28 (2016) 861e876, https://doi.org/10.1007/s10811-
015-0666-6.
[177] F.D. Caprio, P. Altimari, G. Iaquaniello, L. Toro,
F. Pagnanelli, Heterotrophic cultivation of T. obliquus
under non-axenic conditions by uncoupled supply
of nitrogen and glucose, Biochemical Engineering
Journal 145 (2019) 127e136, https://doi.org/
10.1016/j.bej.2019.02.020.

IV. Selected examples and case history