Opinion

Current Bottlenecks and Challenges of the

Microalgal Biorefinery
Imma Gifuni,1,* Antonino Pollio,2 Carl Safi,3 Antonio Marzocchella,1 and Giuseppe Olivieri1,4

Microalgae are increasingly considered as sources of renewable feedstocks for Highlights

industrial production, and microalgae production now focuses on the multi- The exploitation of a single microalgal
product is unprofitable and generates
product microalgal biorefinery. However, such a biorefinery presents several
undesirable waste, inspiring the bior-
bottlenecks that are mainly associated with downstream processes. This efinery approach to microalgae
reduced downstream efficiency results from unsolved problems related to production.

the culture strategy for the accumulation of different products – the protein Several thin culture systems are cur-
versus lipid dilemma – and the dilute nature of the microalgal culture. We rently being proposed for intensifying
identify new trends and propose promising solutions for realizing microalgal the conversion of light into biomass.

biorefineries at industrial scale. New perspectives and challenges are identified Downstream processing, and in parti-
in protein properties and in the integration and cooptimization of culture and cular the fractionation of microalgal
components, remains the most expen-
downstream processes.
sive step limiting the practical imple-
mentation of microalgal biorefineries.

The Microalgal Biorefinery: New Achievements and Unsolved Bottlenecks

The environmental problems of our era have inspired research efforts towards developing new
and sustainable raw resources for food, materials, and energy production. Microalgae are, in
effect, sunlight-driven cell factories that can convert CO2 in chemicals for biofuels, food, feed,
and high-value products [1]. Several companies and start-ups have been founded with the aim
of exploiting microalgal biomass (Microsynbiontix, Cellana, Fermentalg, Microphyt, Algenuity,
a4f, Algafarm, Phytoplancton Marino, and many others), but many studies currently underway
are focusing on how to fulfill the potential of microalgae [2]. Indeed, apart from their advantages,
microalgal cultures are more expensive than heterotrophic fermentation because the culture
step is strongly limited by the extent of light penetration. As a result, cultivation cost scales 1
Dipartimento di Ingegneria Chimica,
linearly with the cultivation area, and photobioreactors (PBRs; see Glossary) are limited by the dei Materiali, e della Produzione
low biomass concentration (
3 g/l for autotrophic microalgal cultures compared to 30–100 g/l Industriale, Università degli Studi di
Napoli ‘Federico II’, Piazzale Tecchio,
for heterotrophic bacteria fermentations), which also affects the harvesting costs.
80, Naples, Italy
2
Dipartimento di Biologia, Università
Significant advantages have been identified in the microalgal biorefinery approach and its degli studi di Napoli ‘Federico II’,
Complesso Universitario Monte
integration into the circular bioeconomy [3]. The biorefinery approach offers a solution for
Sant’Angelo, Via Cinthia, 47, Naples,
profitably exploiting most or all of the microalgal potential through recovering and separating the Italy
3
biomass components as well as by minimizing waste production. In this sense, the microalgal Wageningen Food and Biobased
Research, Wageningen University and
biorefinery cannot only be considered as a set of fractionation steps for the separation of the
Research, PO Box 17, 6700 AA
different products. Instead, the microalgal biorefinery should be considered as a sequence of Wageningen, The Netherlands
4
unit operations that bundle together laboratory-scale research, culture, and downstream steps, Bioprocess Engineering Group,
AlgaeParc, Wageningen University
as shown in Figure 1.
and Research, Droevendaalsesteeg 1,
PO Box 8129, 6708 PB Wageningen,
The current biorefinery approach is still not profitable. The main issue lies in downstream The Netherlands
processes that account for
40% of the total cost [4], mainly because of the dilute nature of the
autotrophic cultures (<3 g/l) and the impossibility of simultaneously optimizing the recovery of *Correspondence:
multiple microalgal components [5]. Thus, solutions to these issues do not come exclusively imma.gifuni@unina.it (I. Gifuni).

242 Trends in Biotechnology, March 2019, Vol. 37, No. 3 https://doi.org/10.1016/j.tibtech.2018.09.006

© 2018 Elsevier Ltd. All rights reserved.
from separately optimizing the operations involved in harvesting and fractionation. The real Glossary
improvement lies in integrating and co-optimizing the different operations (Figure 1). Biorefinery: by analogy to a
petroleum refinery, but where the
Bottlenecks in microalgal biorefineries are present in all operation units. In laboratory-scale raw material is biomass and all of the
components are used in different
research, strain selection, strain engineering, and nutrient supply strategies are currently industrial fields after separation and/
optimized for the hyperaccumulation of a single product. In the culture step, industrial photo- or catalytic conversion.
bioreactors have high costs and reach low biomass concentrations (
3 g/l, while at least a 10- Cascade extraction: a set of
sequential extraction steps aimed at
fold higher concentration is necessary for efficient bioreactor exploitation and downstream
complete fractionation of the initial
operations); accumulation of different products often requires different or opposite cultivation microalgal biomass substrate.
strategies. Finally, downstream processes are designed for a single main product, and the Circular bioeconomy: an economic
remainder is frequently ‘waste’ that must find a destination, with additional cost implications. concept where renewable materials
are used as the feedstock for fuel
and chemical production and all the
This article critically analyzes the key process units of the microalgal biorefinery and focuses on byproducts are completely
some promising strategies to remove the bottlenecks: co-accumulation of marketable prod- degradable.
ucts, an intensified culture system, and recovery of multiple products by a cascade extraction Life cycle assessment (LCA): a
methodology used for estimating and
approach.
assessing the environmental impact
attributable to the life cycle of a
Recent Developments and Promising Solutions product, including climate change,
Culture Strategy: Co-Exploitable Products smog creation, eutrophication,
acidification, toxicological stress on
For decades research studies have focused on single-product exploitation from microalgae. In human health and ecosystems,
this context, most attention has been paid to selecting strains that can best hyperaccumulate depletion of resources, water use,
the target product [6] and to identifying the right nutrient supply and culture conditions [7]. land use, and noise. Generally, a
Interesting results show that microalgae can hyperaccumulate lipids, carbohydrates, and process is analyzed using software,
and international standards are used
carotenoids in nutrient-depleting conditions, especially in the case of nitrogen depletion [8– to calculate the environmental impact
11]. Hyperaccumulation increases the productivity of the target molecule and facilitates its (ISO 1400 series).
recovery from the total biomass [12]. This strategy is considered successful when high-value Photobioreactors (PBRs): closed
reactors used to culture
products are obtained, for example, astaxanthin from Haematococcus spp. or b-carotene from
photosynthetic microorganisms. They
Dunaliella spp. (small market size, 100–1000 s/kg). In these cases, valorization of the remaining can be equipped with different
biomass after the extraction of the main product is conceivable [13]. For species that are known control systems and can have
to accumulate lipids and/or proteins (Scenedesmus spp. and Chlorella spp.), life cycle different shapes. The main feature
that makes a PBR different from a
assessment (LCA) and technoeconomic analysis (TEA) have not concluded that this
simple bioreactor is transparency to
process is economically feasible, especially when low-cost substitutes from fossil sources allow penetration of light.
are considered (biofuel, 0.2–0.5 s/kg) [14]. In these cases, greater benefit can be generated by Polyunsaturated fatty acids
coproducing several medium- and low-value compounds [15]. Maximizing a particular com- (PUFAs): fatty acids characterized
by more than one double bond in
ponent is based on a metabolic imbalance which diverts the electron energy towards the
their structure. They can be classified
selected target and away from other components (further details in Box 1). Hence, it is clear that as methylene-interrupted polyenes
not all the exploitable products are accumulated in the same conditions or growth phase. If the (v-3, -6, -9), conjugated fatty acids,
biorefinery aims to maximize the potential of microalgae, then strategies that consider the co- and other PUFAs. They are widely
recognized for their health benefits
exploitation of different microalgal components should be preferred. From this point of view, and are used in nutraceuticals,
early nitrogen depletion, that characterizes the end of exponential growth phase in a batch cosmetics, and pharmaceuticals.
culture, seems to provide the best compromise for the simultaneous exploitation of polyun- Technoeconomic analysis (TEA):
saturated fatty acids (PUFAs; 15–20 s/kg for food and feed applications) and large market a tool that evaluates available
technology options based on
products such as proteins and starch (2–10 s/kg, food and feed market) [16]. However, technical, economic, environmental,
nitrogen depletion (early or late depletion, Figure 2) is not favorable for growth and biomass social, and regulatory criteria. TEA is
production [17], and further approaches should therefore evaluate two-stage culture (growth in based on the analysis of production
processes by software such as
non-limiting conditions followed by induction of depletion of a substrate) or continuous culture
Aspen to obtain accurate estimates
in limiting conditions at low dilution rate. As reported in some studies, the continuous mode of of mass balance. Models are used to
nutrient limitation is not an effective strategy for increasing the final productivity relative to batch identify reagents and power
or two-stage culture [18]. Moreover, the more practical approach adopted in large-scale requirements. From the flow rates
the cost of each process unit can be
production avoids sophisticated systems and precise control of the dilution rate and nutrient

Trends in Biotechnology, March 2019, Vol. 37, No. 3 243

concentration. These conditions may be difficult to manage, especially in outdoor cultures that estimated and the most significant
are exposed to variable weather and temperature conditions. opportunities for cost reduction can
be identified.
Triacylglycerols (TAGs): esters
Intensive Culture Systems derived from glycerol that contain
The current culture systems adopted for large-scale microalgae production generate very three acyl groups. They are the main
dilute cultures (0.08–3.6 g/l); their productivity is affected by weather conditions and light constituents of fat in both plants
(including algae) and animals
penetration [19,20]. To reduce energy consumption in harvesting and extraction, higher cell (including humans). Their main
concentrations are required (
100 g/l) [21]. This concentration can be achieved only by proposed use is in transesterification
centrifugation, and the energy consumed during the centrifugation step is inversely pro- for biodiesel production (to create
esters of fatty acids); the remaining
portional to the biomass concentration. To reduce the energy costs of centrifugation, a
glycerol is used in food and
common practice is to add a preconcentration step by membrane filtration [22,23], but pharmaceutical production.
preconcentration may be avoided by implementing a thin culture system. These are
currently proposed as a tool for optimizing photosynthetic efficiency, but they can also

Downstream

Nutrients Water and nutrient recycling HarvesƟng/

(N, P, S, ...) disrupƟon
Water

CO2

Culture
Algal FracƟonaƟon
system
strain
Culture

SelecƟon:
Proteins
• Wide range of
Carbohydrates
exploitable products Lipids,
• Most abundant product pigments
• High-value product

Laboratory-scale
research

Figure 1. A Microalgal Biorefinery Integrating Separate Operation Units: Laboratory-Scale Research, Culture, and Downstream Processing.
Laboratory-scale research includes selecting a microalgal strain based on the main target product required and the sources and the supply of low-cost nutrients for
microalgal growth. The culture step concerns the construction, operation, and optimization of a culture system that (i) better fits the microalgal strain requirements in
terms of mixing strategy and light penetration, (ii) reduces the investment and operational costs, and (iii) provides sufficient biomass concentration and productivity for
efficient downstream processing. The downstream unit is composed of (i) harvesting for biomass concentration, (ii) extraction of intracellular molecules by cell disruption
or chemical extraction, and (iii) fractionation of the molecules to maximize the number of commercial products obtained.

244 Trends in Biotechnology, March 2019, Vol. 37, No. 3

 Box 1. Metabolic Imbalance
During autotrophic microalgal growth, CO2 is provided as a carbon source. Hydrogen and oxygen come from water,
while nitrogen, phosphorus, sulfur, and other microelements are supplied as salts [58]. The energy for the synthesis of
organic constituents is captured by the photosystem as electrons from light. The electrons received are distributed in
the synthesis of lipids, starch, and proteins, and some are used in catabolic reactions or are dissipated [59]. Nutrient
depletion can cause metabolic imbalances which can halt growth or redirect the electron flux towards the accumulation
of particular compounds. During stressful conditions (nutrient depletion, high light irradiance, extreme pH, or high
salinity, temperature, or metal concentration), some antioxidant molecules are synthesized by the cells to protect the
organism. These molecules are known as phycobiliproteins, carotenoids, or astaxanthins. In particular, nitrogen level
and growth stage are considered to be the main parameters redirecting microalgal metabolism, and the optimal
conditions for the different product accumulation are reported in Figure 2 in main article. Starch reserves are maximized
during early depletion, while lipids accumulate at late depletion [10]. Different lipid classes also accumulate under in
different conditions: PUFAs require low irradiances at the beginning of stationary phase [16], while triacylglycerols
(TAGs) are produced during the late stationary phase at high light irradiances [60]. The lipid fraction also contains
pigments such as chlorophyll and carotenoids. Chlorophyll captures light for photosynthesis and is mainly associated
with an exponential growth phase, nitrogen-abundant conditions, and low light. Carotenoids are involved in the
response to stress factors and accumulate during nitrogen depletion and high light irradiance [16,53]. In general,
proteins are produced as a result of active cell division, and their production is associated with exponential growth in the
absence of nitrogen or light limitation [54]. A particular class of proteins, the phycobiliproteins (part of the light-harvesting
or ‘antenna’ complex’) reach their maximum concentration at the end of the exponential growth phase, under high light
irradiance and low nutrient availability [55]. Maximum accumulation is related to the specific growth stage and culture
conditions, but there are some phases where the production of stress-associated molecules (carotenoids, lipids,
astaxanthin) is still balanced with a relatively high amount of growth-associated molecules (proteins, chlorophyll). The
best balance regarding the number of exploitable molecules can be found in the late-exponential, early nitrogen-
depletion phase, where proteins, starch, lipids, and secondary pigments coexist.

improve the interconnection with downstream steps, increasing the final biomass concen-
tration (Box 2) with a consequent reduction of processing costs.

Innovative examples of thin reactors have been proposed in the past 5 years. Flat-plate PBRs,
first reported by Qiang and Richmond [24], were improved by reducing the culture thickness

Figure 2. Optimal Conditions for

ExponenƟal Late Early Late
Product Hyperaccumulation Over
phase exponenƟal staƟonary staƟonary
the Growth Phase as a Function
of Nitrogen Concentration. Further
PUFAs TAGs details are given in [10,16,53–57]. Nitro-
Phycobiliproteins gen is provided to microalgae as the key
nutrient for growth. Nitrogen is con-
sumed during growth and is used as a
Starch Carotenoids/ building block to synthesize several
astaxanthin microalgal molecules, most prominently
Chlorophyll proteins. The decreasing nitrogen con-
centration in the medium reduces the
growth of the microorganisms and
causes metabolic imbalances which
Proteins redirect the energy captured by photo-
synthesis towards the production of dif-
ferent molecules (mainly energy reserves
such as starch and lipids, and antioxi-
dants as carotenoids and astaxanthin).
Abbreviations: PUFAs, polyunsaturated
fatty acids; TAGs, triacylglycerols.
Nitrogen- Early Nitrogen Late
replete condiƟons nitrogen depleƟon nitrogen
depleƟon depleƟon

Trends in Biotechnology, March 2019, Vol. 37, No. 3 245

 Box 2. Reducing the Optical Path
Currently, microalgal biomass productivity is limited by poor penetration of light into the culture system. As the biomass
concentration increases in a culture system, mutual shading among the cells establishes a dark zone where photo-
synthesis and growth are prevented. This results in a final biomass concentration of 2–4 g/l. Therefore, the biomass
concentration can be remarkably increased by reducing the optical path of the culture [61,62]. Janssen and coworkers
[59] calculated that by reducing the optical path to 0.01 m it is possible to increase photosynthetic efficiency to up to
5–8%. Thin culture systems, whether open or closed, are innovative solutions to improve light utilization and increase
biomass concentration and productivity [63]. Illustrative examples of thin cultures are shown in Figure I and are
compared to the traditional systems currently adopted. The increased concentration of biomass reduces harvesting
costs.

Thin culture system TradiƟonal culture system

(A) (B)

(C) (D)

Figure I. Examples of Thin Culture Systems and Traditional Culture Systems. (A) Cascade raceways are the
thin version of the (B) traditional open ponds. (C) Thin flat-plate photobioreactors (PBRs) are an innovative architectural
improvement of (D) the tubular PBR. The specifications for these reactors differ according to the production company; in
general terms, the overall biomass productivities are (A) 2–4 kg m3 day1, (B) 0.03 kg m3 day1, (C) 6 kg m3 day1,
and (D) 0.5–1 kg m3 day1. Sources: (A) www.alga.cz; (B) www.seambiontic.com; (C) www.colt.de; and (D) www.
variconaqua.com.

to 3 mm, and these could achieve 24 g/l of biomass (C. sorokiniana) [25]. The flat-plate
system is easy to scale up and operate, and different inclinations allow light intensity to be
modulated. However, one drawback is the power cost necessary to ensure efficient mixing
via bubbling [26]. Integrating a vertical flat panel PBR into a building facade offers oppor-
tunities for efficient energy optimization [27]. Such expensive culture systems can be justified
for high-value products such as astaxanthin and b-carotene (as previously discussed for the
hyperaccumulation strategy). The increase in biomass concentration and the possibility to
induce stress by controlling the operating parameters could effectively improve production.
Alternatively, cascade systems were proposed by Doucha and Lìvanský [28]. They consist of
two inclined cultivation lanes with a culture thickness of 6 mm. This large-scale open

246 Trends in Biotechnology, March 2019, Vol. 37, No. 3

cultivation method achieves cell concentrations between 15 and 35 g/l (Chlorella spp.) [29].
The installation and operation costs are low, but the culture is prone to contamination and
light intensity, temperature, and released CO2 are not controllable. This system is suitable for
lower-value products such as the biomass of Chlorella spp. and Scenedesmus spp. More-
over, these species are considered to be robust and are able to establish themselves as the
main population in open culture. AlgoFilm©, patented by Pruvost and coworkers [30], follows
the same falling-film principle as the cascade system but is completely closed and controlled.
The culture thickness ranges from 1.3 to 2.2 mm. This system represents a competitive
alternative to the closed flat panel for the production of high-value commodities. The highest
biomass concentrations achieved were 17 and 30 g/l in semicontinuous and batch mode,
respectively. The costs of the mixing are reduced with respect to the vertical panel, but are still
not competitive for the production of Chlorella and Scenedesmus for food and feed proposes.
Finally, the foam-bed PBR was published as a patent by Janssen and coworkers [31]. The
thickness of the system is 2.7 cm, but the small amount of liquid culture (5% v/v) and the
formation of foam decreases the depth of the algae layer and increases the surface exposed
to the light. The biomass concentration can reach 10 g/l, but this type of reactor is still far from
industrial scale applications because of the costs and the so far unassessed foaming
technology.

Despite the high biomass concentration achieved in all these culture systems, comparisons
between them depend on whether a closed or open culture system is used. As summarized in
Table 1, the open system is preferred for robust strains (e.g., Chlorella, Scenedesmus,
Nannochloropsis) and is confined to food/feed and fuel applications. Closed and controlled
culture systems offer innovative tools for higher-quality, customized biomass, with applications
in high-value fields (food, pharmaceuticals, cosmetics). The operating costs remain high for this
system, but interesting applications, such as building-integrated PBR, could open the way to
sustainable architecture characterized by attention for the environment, chromatic differentia-
tion, and iconic value [32].

Table 1. Analysis of Advantages, Disadvantages, and Costs of the Different Strategies Reported for Interconnected Biorefinery Unit Operations
Culture strategy Culture system Cascade extraction

Single product Multiple product co- Open thin cascade Thin flat-panel PBR Maximum extraction Multiproduct
hyperaccumulation accumulation raceway of a single target extraction
product

Advantages Simplified recovery Multiple product Low-cost Reduced High purity and Recovery and sale of
of the target product exploitation installation and contamination; high selectivity in the several medium-
operation of the level of control over recovery of the value products
culture culture parameters target

Disadvantages Long depletion time; Complex extraction Contamination; Elaborate Byproduct to low- Mild product
low culture and fractionation variable culture installation and value market (fuel, extraction and
productivity conditions operation energy) multistep
(supporting only requirements for the fractionation
robust strains) culture system

Costs High cultivation cost High profit and lower Low cost High cost for Low cost owing to High cost owing to
owing to long waste (comparable to material and fewer unit mild extraction
starvation time open pond culture) operations operations

Application Astaxanthin by Soluble proteins, Nannochloropsis PUFAs and b-Carotene Proteins, PUFA-rich
(products/strain) Haematococcus lipids, and starch by spp. oil production fucoxanthin by production by oil, and
spp. Chlorella Phaeodactylum Dunaliella salina carbohydrates by
trycornutum Tetraselmis spp.

Trends in Biotechnology, March 2019, Vol. 37, No. 3 247

The Cascade Approach for Multiple Product Recovery
Valorizing the whole biomass in the biorefinery depends on fractionating the multiple compo-
nents and maintaining the yield and the quality of the recovered products. The first step involves
choosing an appropriate extraction method to disrupt the cell wall and release products, and
selecting optimal parameters for selective and mild extraction of the different components
[33,34].

The currently favored approach is cascade extraction. This approach involves sequential
recovery of different microalgal components using consecutive physical and chemical frac-
tionation steps (two-phase extraction, filtration, chromatography, transesterification). The
priority in cascade extraction can vary, and can be the main product of the culture, the value
of the products (high-value products are extracted first), or the sensitivity of the product to the
extraction techniques.

Current studies largely focus on optimizing the recovery of high-value target products (astax-
anthin, b-carotene, lutein), and then employ different methodologies for the valorization of the
remaining byproducts [35–39]. In these cases, the byproduct applications are very limited, and
they cover the production of bioenergy by fermentation of sugars and proteins (mostly dena-
tured) or pyrolysis. This strategy provides added value (waste reduction, further income from
energy production) to production processes which are already justified by the high-value target
product, such as astaxanthin production by Haematococcus spp. Table 1 summarizes
different microalgal culture processes and the different products associated with each strategy.
The table provides a general overview that encourages several combinations of culture
strategies, culture systems, and cascade extractions for different strains and products. When
large-market products for food and tailored feed are considered (functional proteins, edible oil,
starch), extraction of the lipid fraction and valorization of the residue is not considered cost-
effective. Ansari and coworkers [15] examined different scenarios of sequential product
recovery in Scenedesmus obliquus and concluded that the extraction sequence protein–
lipid–carbohydrates gave the maximum recovery yield of all the metabolites simultaneously.
Moreover, if medium- and high-value compounds (such as PUFAs or pigments) are not
damaged by aqueous protein extraction, then protein extraction can precede the recovery
of the high-value compounds. In general terms, the strategy of maximizing the number of
products recovered should consider extraction priority based on the fragility of the molecule,
the use of mild and non-invasive technologies (i.e., low temperature and pressure), and wet-
based extraction for extracting water-soluble components to reduce water evaporation costs.
Water-soluble proteins can find interesting applications as food additives owing to their
functionalities (further explained in Box 3). Microalgae used as sources of proteins for food
and feed can enter the market as whole biomass (e.g., Spirunila, Chlorella, and Tetraselmis
spp.).

Based on these considerations, two different cascade schemes can be proposed (Table 1). The
first, single-product valorization, is adequate for high-value products, such as astaxanthin from
Haematococcus spp. It uses high-yield and cost-effective technology to optimize target
product recovery. In this context, the use of supercritical CO2 assisted by solvents (e.g.,
ethanol) or ionic liquids have been reported to be the most effective approach with low
environmental impact [40]. The exhausted biomass, after the extraction, contains mainly
proteins, sugars, and some residual pigments. The yield and the quality of the residue is
not adequate for the food-additive market, but it can be valorized by producing biofuel via
fermentation or used for feed applications. The second cascade scheme is multiproduct
cascade extraction. Here, robust microalgal strains such as Scenedesmus, Chlorella,

248 Trends in Biotechnology, March 2019, Vol. 37, No. 3

 Box 3. Technofunctional Protein Properties
Proteins play an important role in the production of human food, not only for the supply of essential amino acids but also
for their technofunctional properties in food formulation. The industrial demand for new protein sources, and the rising
prices of soybean and caseinate proteins, have promoted research interest in microalgal proteins with favorable
properties.

Emulsifying Properties

Emulsification can be promoted by specific proteins that play a key role in the formation and stabilization of an emulsion.
Several studies have investigated the emulsifying capacity of proteins extracted from numerous microalgal species
[64,65]. The emulsion capacity (EC) and emulsion stability (ES) of the proteins are linked to several factors: the
technology used for extraction, the solubility and the composition of the protein isolate, the isoelectric point, the
species selected, and the pH. Proteins isolated from Haematococcus pluvialis and Chlorella vulgaris have shown
promising emulsifying properties [47,64].

Foaming Properties

A foam is defined as a two-phase system that consists of an air volume coated by a very thin layer of liquid. Two
important foaming properties are foam capacity (FC) and foam stability (FS). Similar to emulsification, foaming is affected
by several factors such as the physiological characteristics of the species and the pH. Arthrospira platensis proteins
showed the highest foaming capacity and stability at pH 10 [66,67], while the best foaming performance was observed
at pH 7 for species such as Tetraselmis [68] and Nannochloropsis gaditana [69] that have tough cell walls.

Gelling Properties

Gel formation occurs when partially unfolded proteins develop uncoiled polypeptide segments that interact at specific
points to form a 3D crosslinked network [66]. Partial unfolding of functional proteins is mainly due to heat and pH
(pH  5; pH  11). Chronakis [70] reported the possibility of obtaining suitable gelling properties from Spirulina platensis
proteins at low protein concentration (1.5–2.5 wt%) at 90  C and with CaCl2 as a stabilizer.

Antioxidant Activity

Microalgal proteins or peptides can prevent the harm caused by oxidative stress. Antioxidants reduce or neutralize the
oxidation process that generates cell-damaging free radicals. Peptides derived from the enzymatic hydrolysis of C.
vulgaris protein residues can scavenge several free radicals [51]. Furthermore, phycocyanin – a phycobiliprotein found in
cyanobacteria such as S. platensis – also features antioxidant properties [71,72].

Tetraselmis, Phaeodactylum, and Nannochloropsis spp. are employed. The molecules pro-
duced vary from medium value (PUFA oil-rich, functional proteins) to low value (starch or
insoluble proteins). An innovative cascade scheme may consist of a first extraction of water-
soluble components (mainly proteins and sugars) by mild disruption and centrifugation,
followed by recovery and concentration of proteins for the food market. The second extraction
step should be lipid extraction, and in some cases separation and enrichment of particular lipid
classes (such as PUFAs). The last step considers the recovery and the valorization of the starch
fraction in the residue for application in bioplastic production.

In a multiproduct cascade, coextraction generally occurs because of similar physical or chemical

properties, and further separation of the mixture is therefore not an easy task and additional costs
may be involved. ‘t Lam and coworkers [41] related the economic feasibility of the fractionation of
multiple products to the number of unit operations. They proposed the use of combined process
units to reduce the cost, such as the use of ionic liquids for one-step extraction of hydrophilic and
hydrophobic molecules [41]. The present work highlights the possibility of introducing new
operation units into the fractionation step if TEA demonstrates that most valuable products
are to be recovered, leading to increased profit. Moreover, the optimization proposed here is
not related to a single fractionation step but to optimizing interconnections among the different

Trends in Biotechnology, March 2019, Vol. 37, No. 3 249

operation units (culture strategy, culture system, downstream). The optimal strategy is always Outstanding Questions
related to the strain and the main target product (Table 1), but as sustainable sources of natural What is the right culture strategy for
products, microalgal biorefineries should always consider eco-friendly techniques. microalgal biorefineries?

Is it worth investing in optimizing thin

The Challenge of New Microalgal Products photobioreactors?
An important reason that multiproduct microalgal biorefineries are not cost-effective is because
many microalgal products have not yet been explored. For instance, the residual fraction of the Should we invest in minimizing the
second cascade scheme previously mentioned culminates in insoluble components such as fractionation process costs or in maxi-
mizing the number and quality of the
starch, proteins, and ash. This mixture could be valorized for bioplastic applications, or as a food
products recovered?
additive or emulsifier, because of its high starch content [10,42]. In addition, depending on the
separation techniques used to extract the target product, a proportion of high-value molecules Are there any added-value fractions
(functional proteins, antimicrobials, or antioxidant compounds) may be lost in the residual fraction. that have not yet been valorized in
microalgal biomass?
One rapidly increasing area of interest in sustainable protein sources is to incorporate more plant-
based proteins in food/feed in place of animal-based proteins [43]. Microalgae are an abundant
source of proteins owing to their ability to accumulate significant amounts of proteins and essential
amino acids. Their potential use as feed, and in particular as aquafeed nutrition, has been
emphasized [44], but new and interesting applications are emerging. Therefore, it is worthwhile
to explore and evaluate the potential of their functional properties, for example, as emulsification,
foaming, and gelling agents, or as antioxidants (Box 3). Recent studies have demonstrated that
microalgal proteins have similar or better properties than standard protein sources such as whey
proteins [45], soybean flour [46], and egg protein [47]. Current obstacles to using microalgal
proteins in the food industry include the difficulty of separating protein–chlorophyll complexes
(which have intense color and taste) without altering the protein functionality [48], and preserving
protein functionality during cell disruption and cascade extraction [49]. Thus, more attention
should be paid to the use of mild and non-invasive technologies for microalgal biomass fraction-
ation. Promising results have been reported by Safi and colleagues [50] concerning ultrafiltration
technology for purifying these proteins. Further, Suarez Garcia and coworkers [45] demonstrated
that lower protein purity in crude bead milling extract ensures optimal foaming, emulsifying, and
gelling properties. About 1 kg of oil is emulsified per gram of microalgal protein-rich extract, and
75% stability is ensured. Moreover, extracts containing only 3% algal proteins show gel-forming
properties, whereas extracts containing 10% protein are required for gel formation by whey
proteins. Nevertheless, further characterization of microalgal proteins is required, as well as
strategies to integrate their recovery in the biorefinery context. Furthermore, the strong relationship
between the structure and function of the proteins and their sensitivity to several factors (including
pH, temperature, solvents, salt concentration, and the presence of bacteria) suggest the that ad
hoc protocols will need to be developed for their storage and to preserve their functionality during
large-scale processes.

Another property that can be valorized in the microalgal biorefinery is the antioxidant and
antimicrobial activity of several fractions, including proteins and peptides. Frequently, a residual
fraction containing hydrolyzed proteins, that has no evident technofunctional properties, could
contain antioxidant or antimicrobial activity [51,52]. Greater attention should be paid to the
characterization and valorization of residues after extraction of the main product.

Concluding Remarks
Significant improvements have been made in the field of microalgal technologies. Some
examples are the biorefinery approach, the development of thin culture systems, and the
study of cascade extraction for multiple product fractionation. An optimal microalgal biorefinery
at industrial scale needs to focus on the cointegration and co-optimization of the different

250 Trends in Biotechnology, March 2019, Vol. 37, No. 3

operation units (see Outstanding Questions). Thin (open or closed) culture systems may
approach the required concentrations for both disruption and fractionation steps. The optimal
strategies for upstream, culture, and downstream processing mainly depend on the strain and
the target product.

Following this optimization, two biorefinery approaches can be defined for either high-value
products or medium/low-value products. The first should use a hyperaccumulation strategy
and a closed thin flat PBR to maximize target product extraction and valorize the byproducts.
The second should prefer the late exponential phase in early nitrogen-depletion as the best
phase for the co-accumulation of several exploitable products, and use a thin cascade raceway
and a multiproduct cascade approach in which extraction priority is based on the fragility of the
recovered compounds (soluble proteins ! pigments/lipids ! starch). The cost of introducing
complex and expensive technologies for further fractionation must be mitigated by real
economic advantages. Finally, it is important to valorize all the microalgal components, such
as soluble proteins, which have functionality as food additives instead of as mere protein
sources for food and feed. In addition, new properties may be present in the residual fraction
after the main compounds have been extracted, and these need to be investigated for their
potential to create new commercial products.

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