Biomass From Microalgae: The Potential of Domestication Towards Sustainable Biofactories

Benedetti et al.
Microb Cell Fact (2018) 17:173

https://doi.org/10.1186/s12934-018-1019-3 Microbial Cell Factories
REVIEW Open Access
Biomass from microalgae: the potential

of domestication towards sustainable
biofactories
Manuel Benedetti, Valeria Vecchi, Simone Barera and Luca Dall’Osto*
Abstract
Interest in bulk biomass from microalgae, for the extraction of high-value nutraceuticals, bio-products, animal feed
and as a source of renewable fuels, is high. Advantages of microalgal vs. plant biomass production include higher
yield, use of non-arable land, recovery of nutrients from wastewater, efficient carbon capture and faster development
of new domesticated strains. Moreover, adaptation to a wide range of environmental conditions evolved a great
genetic diversity within this polyphyletic group, making microalgae a rich source of interesting and useful metabo-
lites. Microalgae have the potential to satisfy many global demands; however, realization of this potential requires
a decrease of the current production costs. Average productivity of the most common industrial strains is far lower
than maximal theoretical estimations, suggesting that identification of factors limiting biomass yield and removing
bottlenecks are pivotal in domestication strategies aimed to make algal-derived bio-products profitable on the indus-
trial scale. In particular, the light-to-biomass conversion efficiency represents a major constraint to finally fill the gap
between theoretical and industrial productivity. In this respect, recent results suggest that significant yield enhance-
ment is feasible. Full realization of this potential requires further advances in cultivation techniques, together with
genetic manipulation of both algal physiology and metabolic networks, to maximize the efficiency with which solar
energy is converted into biomass and bio-products. In this review, we draft the molecular events of photosynthesis
which regulate the conversion of light into biomass, and discuss how these can be targeted to enhance productivity
through mutagenesis, strain selection or genetic engineering. We outline major successes reached, and promising
strategies to achieving significant contributions to future microalgae-based biotechnology.
Keywords: Microalgae, Photosynthesis, Photobioreactor, Biomass, Bio-based products, Light-use efficiency, Strain
domestication, Molecular genetic
Background storing it efficiently. Oxygenic photosynthesis converts

Microalgae, a promising feedstock option CO2 into reduced carbon compounds using light and
Approximately 100,000 terawatts-year (TW-y) power water; through this process photoautotrophic organ-
from sunlight reach the surface of our planet. This is a isms, namely plants, algae and cyanobacteria, store solar
renewable resource exceeding the current human global energy at a rate of 120 TW-y at the global scale. There-
energy demand (15 TW-y) and the 24 TW-year pre- fore, using sunlight and C O2 to produce a variety of
dicted for anthropic activities by 2030 [1, 2] by > 3 orders organic molecules and biomass, by the extensive culti-
of magnitude. Sunlight might fully provide for future vation of photosynthetic organisms, has the potential to
world energy demand [3] and yet its dilute nature rep- cover a significant portion of global energy demand [4],
resents a major challenge for concentrating, harvesting, besides providing for an effective CO2 capture from e.g.
power plants or other large-scale emission sources. As
*Correspondence: [email protected]
unique feature, photosynthesis allows for direct energy
Dipartimento di Biotecnologie, Università di Verona, Strada Le Grazie 15, storage into liquid fuels which can be used in the existing
37134 Verona, Italy
© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Benedetti et al. Microb Cell Fact (2018) 17:173 Page 2 of 18
transport systems, while other forms of renewable energy Multiple reasons contribute to such a feature:
such as hydro-, wind or photovoltaic power, cannot.
Thus, mass culture of microalgae has gained inter- i. When growing in aerated liquid cultures, cells have
est in the past few decades. Indeed, beside small-scale easy access to light, CO2 and nutrients. They lack
traditional cultivations mainly aimed to human feeding, non-productive (heterotrophic) organs to main-
commercial production of algae on a larger scale has tain, and the simpler unicellular structure make the
been identified in recent years as a renewable and envi- whole biomass fully photosynthetically active, irre-
ronmentally sustainable strategy for feedstock produc- spective to seasonal life cycle;
tion. Microalgae include a wide group of photosynthetic, ii. Algae are metabolically flexible and have a short
eukaryotic, unicellular organisms: green microalgae, doubling time. Although most microalgae are pri-
belonging to the class Chlorophyceae, include genera marily photoautotrophs, many species undergo
which are among the most widely used for industrial metabolic shift to heterotrophy upon changes in
applications, such as Haematococcus, Chlorella and environmental conditions, utilizing organic com-
Dunaliella. Diatoms and cyanobacteria, which also rep- pounds as C and energy source or to mixotrophy
resent a valuable biotechnological platform [5, 6], will not (carrying out photosynthesis as the main energy
be included in this review. source, and both organic molecules and carbon
The phyletic group of green microalgae include spe- dioxide are used as C source). Moreover, under
cies which have adapted to diverse environmental con- optimal growth conditions, most species have dou-
ditions, even extreme, of the planet [7]. By considering bling time of a few hours, and cultures reach as
that the unclassified species likely represent the major- much as 10 g l−1 of heterotrophic dry weight (DW)
ity of this group [8], it comes that green microalgae are biomass and 6 g l−1 of photoautotrophic DW bio-
a source of metabolic and genetic diversity [9]. Microal- mass [22, 23];
gal biomass represents an energy-rich feedstock, which iii. Microalgae do not require fertile land, and can
received increasing attention for commercial cultivation grow in wastelands, using brackish or waste water,
in open ponds or closed photobioreactors (PBRs). So far or even sea water in the case of marine species;
industrial applications include production of bioactive hence, their cultivation does not compete with
compounds [10], recombinant proteins [11], next genera- resources for conventional food production, and
tion biofuels and wastewater treatment [12]. Once target would be more environmentally sustainable respect
products extracted, the residual biomass can be further to extensive cultivation of crops. Therefore, micro-
processed into livestock feed, organic fertilizer and algae offer the opportunity to shift part of unsus-
biostimulants, or used for energy cogeneration [13–15]; tainable farming and fishing routines toward
therefore, biorefinery processes applied to mono-species unproductive region;
cultivation can yield a large variety of resources. iv. Different species can be selected for specific
Although photosynthetic machinery is similar to that growth conditions, suited to the local climate,
of plants, microalgae convert solar energy into biomass which is more difficult with conventional crops.
and fix CO2 at efficiencies that are appreciably higher
than land plants [16]. The maximal conversion efficien-
cies of solar radiation into biomass are 4.6% for C3 plants Main text
and 6.0% for C4 plants at 30 °C, which drops to 2.9% and The most promising microalgae species for production
4.2% respectively, when measured in the field [17]. of valuable compounds and for biotechnology applications
Theoretical quantification of 8–10% in energy con- In this paragraph, the bio-technological applications of
version efficiency of microalgae [18] translates into an best wild type species are reviewed with focus on high-
expected maximal productivity of 280 ton of algal bio- value production chemicals and biomass for biofuels
mass hectare (ha)−1 year−1, while outdoor mass cultiva- (Table 1). Currently, the most relevant microalgal species
tion record yield beyond 100 ton ha−1 year−1 could not be for high-value chemicals production are the cyanobacte-
reached [19]. This compares to 0.2% of energy conversion rium Arthrospira platensis (formerly known as Spirulina)
efficiency and an average of 10 ton ha−1 year−1, reported and the green microalgae Chlorella vulgaris, Dunaliella
for sugarcane field trials in the tropics. When considering salina and Haematococcus pluvialis, which are mainly
oil yield extracted from plant vs. algal biomass, palm oil dedicated to the production of single products in large-
can produce a maximum of 4–5 ton ha−1 year−1 [20, 21] scale cultivation systems.
vs. 30 ton ha−1 year−1 [19]. Thus, record yields of micro- Arthrospira platensis is exploited as source of nutra-
algal culture at temperate latitudes is > 5 times higher ceuticals [24], long-chain polyunsaturated fatty acids
respect to the case of the best figure for a plant crop. (lc-PUFAs) [25], carotenoids [26] and proteins [27].
Table 1 Noteworthy microalgae species and their biotechnological applications for production of high-value metabolites
Application Industrial Cosmetics and food High-value Biofuel Dietary supplement
and medical field colorant metabolites and nutraceuticals
Bioproduct category Polysaccharides Phycobiliproteins Mycosporine-like amino Oil to biodiesel Polyunsaturated fatty
Microalgae species Chlorella spp. Arthrospyra platensis acids Botryococcus braunii acids (PUFAs)
(metabolite) (β-glucans, starch) (phycocyanin) Aphanizomenon flos- Chlorella spp. Parietochloris incise
Porphyridium cruentum Carotenoids aquae Dunaliella salina Porphyridium spp. (ara-
Netrium digitus Arthrospyra platensis Vitamins Monoraphidium chidonic acid)
Phycotoxins Chlorella vugaris Euglena gracilis (biotin, contortum Arthrospyra platensis
Amphidinium Haematococcus α-tocopherol) Scenedesmus spp. Rhodomonas salina
Dinophysis pluvialis Prototheca moriformis Carbohydrate to Tetraselmis uecica
Prorocentrum Chlorella zofingiensis (ascorbic acid) bioethanol (α-linolenic acid)
Phycobiliproteins (astaxanthin) Arthrospyra platensis Spirogyra spp. Chlorella minutissima
Red algae (Phycoeryth- Dunaliella salina Chlorella spp. Chlorococum spp. Monodosus spp.
rin) (β-carotene) Proteins Bio-hydrogen Nannochloropsis spp.
Arthrospyra platensis Chlamydomonas Neochloris oleoabundans
Chlorella spp. reinhardtii Pavlova lutheri (eicosap-
entaenoic acid)
Crypthecodiuimu spp.
Isochrysis galbana
Schizochytrium spp.
Thalassiosira spp.
Thraustochytrium spp.
(docosahexaenoic acid)
Other applications of Spirulina are in the medical field Astaxanthin is a high-value, red keto-carotenoid, suc-
as a therapeutic [28, 29], as antioxidant [25] and for the cessfully commercialized by many companies worldwide
extraction of the blue pigment phycocyanin, approved through cultivation of the green alga Haematococcus plu-
as food colorant by FDA [30]. vialis. Under various stress conditions, this alga changes
Genus Chlorella includes a number of species which from a thin-wall mobile phase to a red thick-wall resting
are widely commercialized for production of nutra- phase, in which astaxanthin can reach up to 5% DW [38].
ceuticals. Besides to the high protein, carotenoids and Astaxanthin is widely employed in the feed, cosmetic,
vitamins content [31], C. vulgaris contains also β- and aquaculture, nutraceutical and pharmaceutical indus-
α-glucans, d-glucose polysaccharides which act as tries because of its antioxidant potential [39]. Moreover,
immune stimulators, free-radical scavengers and anti- astaxanthin-rich Haematococcus is a popular nutraceuti-
cancer compounds [32]. Moreover, Chlorella has been cal antioxidant for human diet [40]. Chlorella zofingiensis
successfully used to produce starch, reaching 26% DW has been proposed as an alternative astaxanthin source
yield under mixotrophic condition [33]. which is more reliable in growth [41].
Carotenoids represent the commercial product from Biosynthesis of fatty acids and triglycerides (TAGs)
microalgae with highest success. Carotenoids are is relevant for several industrial applications. Micro-
widely used as food colorants, aquaculture feed addi- algae are the primary producers of lc-PUFAs such as
tives and components for cosmetics and skin care; eicosapentaenoic acid (EPA) and docosahexaenoic
carotenoids also have biomedical applications, includ- acid (DHA), which accumulate in the oil of many fish
ing anti-inflammatory activities which are related to species. Aquaculture farming increased demand of lc-
their strong antioxidant properties [34]. β-carotene, the PUFAs for nutrition, which are currently produced
first carotenoid successfully marketed at large scale, is from fish oil, while a more sustainable lc-PUFA supply
produced from the halophilic alga Dunaliella salina is seeked. Several marine algal species are rich in lc-
through both extensive cultivation in ponds and inten- PUFAs thus have a great potential for biorefinery: these
sive cultivation in PBRs [35]. Recently, new strains with include Thraustochytrium sp., Pavlova lutheri, Nanno-
different ability to accumulate carotenoids and differ- chloropsis gaditana, Isochrysis galbana, Crypthecodin-
ent capacity of photoprotection against high light stress ium cohnii (rich in DHA and EPA), Rhodomonas salina
have been isolated; the most promising strain was char- and Tetraselmis suecica (α-linolenic acid) [42, 43] and
acterized by a β-carotene productivity of 3.5 g l−1 day−1 Parietochloris incisa (arachidonic acid) [44]; these are
at 1500 µmol m−2 s−1 [36]. Currently, various strains currently exploited by a number of small companies,
of D. salina growing at different salinity conditions are marketing biomass of high-values but at small-scale.
available [37]. Lc-PUFAs are important elements for human diet:
DHA plays a crucial role as anti-inflammation molecule tested, including cell wall disruption by chemical/physi-
in allergic diseases and has considerable benefits on cal methods or enzymatic hydrolysis, which enhanced
visual and cognitive functions; optimization of the ratio bio-methane yield [14, 61, 62].
of lc-PUFAs in nutraceuticals may contribute to reduce Finally, microalgae and cyanobacteria can produce bio-
the severity of allergic disease symptoms [45]; oil from hydrogen through photo-fermentation, in an anaerobic
Nannochloropsis, Rhodomonas and Tetraselmis has process involving oxidation of ferredoxin by the hydroge-
higher antioxidant properties respect to fish oil, likely nase enzyme [63, 64]. Although biological H2 shows great
due to the content of valuable carotenoids and polyphe- promise for generating future, large scale sustainable
nols and is expected to replace fish oils in diets soon energy, a number of bottlenecks still limit its production
[46]. [65]; however, recent result [66] identified in C. rein-
Further species with high potential for large-scale hardtii promising targets for genetic engineering of H 2
exploitation of extracellular polysaccharides include production capacity while the use of temperature-sensi-
the microalgae Porphyridium and Desmidiales spp. Red tive conditional PSII mutants has been proposed in order
microalgae Porphyridium spp. are fast-growing and accu- to separate the oxygenic biomass-accumulating phase
mulate extracellular polysaccharides commercially used from the oxygen sensitive hydrogenase activity [67].
in cosmetic and medical field [47, 48]. Amongst Desmidi- Microalgae are gaining importance in the biologi-
ales, Netrium digitus has been successfully cultivated in cal offset of polluted matrix, because of their ability
porous substrate bioreactor, reaching a maximum prod- to thrive under extreme or polluted condition: they
uct concentration of 25 g m−2 [49]. serve for direct carbon capture, a way for reducing CO2
In the last decade, microalgae have received increasing released by large-scale emission plants [68]. Promis-
interest as a source of biomass for replacing fossil fuels. ing species include Scenedesmus obliquus, Chlorella
Liquid fuels derived from raw biomass are an attractive vulgaris, Chlorella protothecoides and Spirulina spp.,
source of renewable energy, to be used in transport sys- which can grow up to 15–18% CO2, although highest
tem or energy cogeneration. With respect to the major productivity was observed around 10% C O2 [69, 70]. In
biofuels currently produced worldwide, namely bio-eth- last years, N. gaditana is arising as promising species for
anol from sugar cane and biodiesel from oil crops, the so CO2 removal due to a high biofixation rate—more than
called “third generation” (microalgal) biofuels, are con- 1.7 g−1 l−1 day−1 [71].
sidered as a promising option, since these organisms are Growth in open ponds is an established technology for
highly productive and provide a solution for the food vs. bioremediation of wastewater and nutrient recovery in
fuel problem [50]. the form of biomass [12]. The effectiveness of microal-
Green algae accumulate high levels of polysaccharides gae to use inorganic N and P to sustain growth as well as
both as cell-wall constituents and storage molecules that their capacity to sequester heavy metals and toxic com-
can be fermented to bioethanol [51, 52]. Oil fraction of pounds, have been demonstrated with a wide range of
algal biomass, which range from 20 to 60% DW depend- wastewaters, and at a range of scales [72, 73].
ing on the species and growth conditions [19, 53], is pro-
cessed by transesterification to produce biodiesel. In this Technical challenges to cost‑effective, large‑scale
respect, promising species belong to the genus Chlorella microalgae production
[54], Scenedesmus [55] and Monoraphidium, the latter Despite a number of industrial applications of micro-
showing high productivity and high-quality lipid pro- algae have been proposed and studied in lab-scale, the
file [56]. An unusually rich source of TAGs is the green only successful commercial exploitation of microalgal
microalga Botryococcus braunii (hydrocarbons constitut- mass culture is the production of carotenoids, namely
ing up to 75% of its DW), however its potential is limited β-carotene by D. salina, and astaxanthin by H. pluvialis
by the slow growth [57]. [10]. Other species (Chlorella spp., Spirulina spp.) pro-
Despite great advantages offered by microalgae exploi- duce high-value compounds, however these productions
tation, production of third generation biodiesel is still are currently applied at small-scale cultivations, which
far from being commercially viable [58]. As an alterna- need significant reduction in operating costs to become
tive, biogas generation through anaerobic digestion of competitive with the same molecules extracted from
microalgal biomass has been proposed as a more ener- other feedstocks [74]. A number of species have been
getically-favorable process [59]. The efficiency of biogas identified as promising targets for biorefinery approaches
production is species-dependent because is based on cell [75], which, however, have not yet come to economic
wall degradability and sometime limited by the content viability. Thus, while microalgae represent a promising
in molecules inhibiting growth of methanogenic Archaea source of valuable bio-based products, (1) optimization
[60]. A number of pre-treatment procedures have been of both cultivation and processing technologies, together
with (2) selection of candidates with high growth rate procedures: electrolytes or polymers are added to floc-
and cell density, are required to make the process profit- culate cells; centrifugation or flow filtration are rapid
able [76]. An overview of the approaches and the major methods, which however implies high investments
challenges related to point (1) are presented as follows. and operating costs; biological-based methods include
In algal biomass pipeline, there are many elements auto-flocculation (at high pH, in excess of C a2+ ions),
which contribute to the overall cost of the process. Major bio-flocculation (caused by secreted polymers) or micro-
factors to take into account are (i) the choice of pro- bial-induced flocculation [83]. Dewatering and drying of
duction system, (ii) the strategies to supply nutrients, biomass is required, and the low biomass concentration
aeration, mixing, and (iii) how to harvest and process (0.1–1% w/w) affects the cost of the final product [84]. In
biomass, and (iv) the procedure to avoid infections and case the release of the products from cells is required, it
contaminants. Different approaches are available, each should be as more energy-efficient as possible, avoiding
having benefits and limitations [77]. the use of expensive solvents, and costs for treating bio-
Microalgae are mainly cultivated in open ponds, which mass should be minimized. Novel approaches which limit
are cheaper to build, and easier to operate and to scale- the use of solvents, e.g. based on enzymatic hydrolysis
up than closed systems. Generally, ponds are raceways at of the cell wall [51, 61], still suffer for expensive enzyme
depth of 20–30 cm, in which biomass is mixed by pad- production. To significantly reduce energy penalty of the
dles or left unstirred. Drawbacks of these systems include production process, the waste biomass can be directed to
the complication of controlling contaminations, and the anaerobic digestion and production of biogas, fertilizers,
difficult of keeping constant growth parameters (e.g. soil amendments or feeds [13, 62, 85].
temperature, pH, light); moreover, they suffer of low pro- Figure 1 shows the different stages in the production
ductivities (< 20 g m−2 day−1) due to poor gas exchange of algal biomass, including the factors to be considered
and dark zone, therefore low cell density forces to cover and optimized, which contribute to the price of bio-
extensive areas and requires high costs for harvesting the products. In consideration of the processing costs at the
biomass. present state of technology, engineering optimization is
As an alternative to open ponds, closed PBRs allow necessary to find new, cost-effective methods of produc-
for higher productivities (~ 0.8–1.5 g l−1 day−1 [19], up ing large quantities of feedstock. However, integration of
to 10 times higher than ponds). Lab-scale PBRs include innovative technical solutions with strains improved by
flat reactors, tubular reactors or vertical plastic bags, with biotechnological approaches, appears essential.
more control over the growth environment, in which The overall cost of a biomolecule is the results of cellu-
biomass is mixed by air-lift or by pumping. These con- lar content of the desired product, and growth rate of the
figurations allow higher cell density than ponds, thus culture, the latter being dependent on the efficiency at
improve economic viability of production, however they which photosynthetically active radiation (PAR) is used
(i) have high building costs and are difficult to scale-up, to drive photosynthesis. Indeed, an area of promising
(ii) can operate in a sterile mode, which however adds research aims at improving the light-to-biomass conver-
to the management fees, and (iii) require a high energy sion efficiency under mass culture conditions.
input for gas exchange [78, 79]. Together with light, CO2
and nutrients must be supplied to maximize the growth
rate, and it significantly affects the economy balance. CO2 Biological constraints in light‑to‑biomass conversion
can be delivered through direct bubbling, and its distri- efficiency
bution in the culture represents an additional cost fac- Calculations in [18] provided both theoretical maxima of
tor; another challenge is the removal of excess O 2 which, solar energy conversion efficiencies in photosynthesis and
above air level, inhibits photosynthesis [80]. productivity yield of microalgae, equal to 8–10% solar-to-
A third production method is the surface-attached algal biomass and 280 ton of dry biomass ha−1 year−1, respec-
biofilm, which showed greater yield than suspended cul- tively. Instead, outdoor mass cultivation showed that,
ture, and lower land and water requirements, in lab-scale with the present technology and wild type strains, annual
trials. Algal biofilm system thus appears a good option productivities beyond 80–100 ton DW ha−1 year−1 can-
for low-cost productions [81, 82], however more research not be maintained at large scale and over long periods
is needed to move from bench-scale to pilot plant. [19]. Overcoming this gap, which limits exploitation of
Following growth, biomass must be (i) harvested and microalgae to their full potential, is therefore essential.
(ii) processed to dryness. Both steps remain a major Wild type algal strains suffer of light use inefficiency.
obstacle to industrial scale processing and contribute Enhancing light-to-biomass conversion efficiency will
to ~ 1/3 of the final biomass cost. Current harvesting help counterbalancing the cost of energy and nutrients
methods include chemical, mechanical and bio-based used in the cultivation system, as well as reducing the
Fig. 1 General scheme of the algal production chain. A number of factors, including the high cost of the infrastructure and the energy required for
growth, harvesting and processing the algal biomass, significantly contribute to the cost of the whole production pipeline
costs of downstream biomass processing, making it a tar- a feedback-regulated de-excitation of Chls that operates
get for genetic improvement. in the PSII, to prevent over-excitation of reaction cent-
Regulation of light harvesting capacity is crucial for ers (Fig. 2). Although light-dependent energy quench-
cells in order to balance light reactions and downstream ing is a property of all photosynthetic organisms, large
biochemical events of photosynthesis. Indeed, auto- differences in amplitude and kinetics can be observed.
trophs have evolved regulatory mechanisms, to fine-tune Some microalgae, such as C. zofingiensis, exhibit con-
continuous transitions between “conservative” and “dissi- stitutively high energy quenching activity [86], while in
pative” state of absorbed energy. In particular, photosyn- other species (e.g. C. reinhardtii) energy quenching is
thesis typically displays a light saturation curve (Fig. 2), significantly activated only upon acclimation to excess
in which 3 distinct phases can be identified: (1) at low light conditions [87]. NPQ activates when light excita-
irradiance, namely when light is the limiting factor, the tion flux exceeds CO2 fixation rate. However, algae can
photosynthetic rate increases linearly with light intensity; experience very high light intensity, saturating photopro-
(2) at increasing irradiances, the limiting factor becomes tective mechanisms. Light in excess of photosynthesis
CO2 fixation rate, thus photosynthetic rate increases saturation level is dissipated rather than contributing to
non-linearly as a function of light; (3) when light inten- biomass accumulation, or even causes synthesis of reac-
sity overcomes the rate of downstream biochemical reactive oxygen species (ROS), which damage the photosyn-
tions, photosystems get rid of energy absorbed in excess, thetic machinery and affect biomass yield. C. reinhardtii
and in this phase photosynthetic rate reaches a plateau. mutant npq4, devoid of NPQ response, was indeed more
In a dilute culture of C. vulgaris, where light attenua- susceptible to photo-oxidation [87].
tion is minimized, light saturation is reached at around Due to the absorption of pigments bound to the large
1200 µmol photons m−2 s−1. At this irradiance, algae pro- antenna systems in both photosystems, light distribution
tect themselves from excess illumination by triggering within the culture is inhomogeneous, and this strongly
the non-photochemical quenching (NPQ) mechanism, contributes to the gap between theoretical vs. real
Fig. 2 Light response curves for photosynthesis. The light compensation point is the minimum light intensity at which the organism shows a gain
of carbon fixation. The net photosynthetic rate shows a linear rise in response to increased light, in the range of light limitation. At higher light
levels, saturation occurs as the efficiency of the photosynthetic mechanism is reduced due to the activation of energy quenching processes. Under
excess light conditions, net photosynthesis can decline as a result of photoxidative stress
productivity. Such large arrays of antenna complexes have culture limits shading while maximizes light absorption
been selected by evolution as favorable trait, since they and net photosynthesis.
maximize the light-harvesting capacity and therefore the One additional factor which contributes to the ineffi-
adaptation to a natural water environment where light is ciency of photosynthesis is carbon fixation: the enzyme
often scarce and limits growth, and cell density remains RuBisCO, which catalyzes the carboxylation of Ribulose
low. Contrary to the natural environment, growth con- 1,5-bisphosphate (RuBP), has low affinity for C O2 and can
ditions in mass cultures requires high cell biomass per also use O2 to give oxygenated substrates [90], which ulti-
volume of installed facility. However, this results in high mately results in ATP/NADPH consumption and loss of
optical density and light shortage in the deeper layers of fixed C. To compensate for this, RuBisCO accumulates to
the culture while cells at the surface layers intercept most as much as 50% of total soluble proteins of the cell. Inef-
photons, resulting in saturation of photosynthesis, dissi- ficient light use in mass culture, due to inhomogeneous
pation of excess energy and/or photoinhibition. The most light distribution, results from limitations in the turnover
inner layers easily drop below the compensation point of rate of the Calvin-Benson cycle. The selection of strains
photosynthesis while active respiration consumes energy. with higher RuBP carboxylation activity would therefore
Thus, dense algal cultures suffer both photo-deprivation be a major goal for the optimization of photosynthesis.
and photo-inhibition, decreasing the overall light-to- Possible strategies include the heterologous expression of
biomass conversion efficiency far below the theoretical variants of RuBisCO with a higher specific activity, or the
score. Rapid mixing of biomass is often suggested as a control of allosteric regulators (e.g. RuBisCO activase) to
solution to light gradients, but it is not: rapid light/dark yield into suppression of oxygenase activity.
cycles between dark and over-saturating irradiances have Interestingly, many microalgae use biophysical carbon
a deleterious effect on biomass yield [88]. Modeling of concentrating mechanisms for active retention of inor-
the light-response curve of photosynthesis in a culture ganic C [91] to increase CO2 availability at the RuBisCO
system [89] suggests that optimal setting of OD in the active site within the pyrenoid, a micro-compartment of
the chloroplast. Engineering such a mechanism into algal proposed to perform better in light transmittance than
species devoid of pyrenoids might augment the overall C wild type [95]. Mutagenesis and screening of C. rein-
fixation rate and thus photosynthetic efficiency. hardtii was employed to isolate mutants having a trun-
In conclusion, results reported in this section suggest cated light-harvesting system [96–100]: all of them
that modulation of photosynthetic reactions is a key fac- showed a higher productivity than the wild type in
tor controlling biomass yield at both saturating and sub- bench-scale growth systems. Cazzaniga and collabo-
saturating irradiances, that is worth to be considered rators [101] applied random mutagenesis to a ther-
for a domestication strategy aimed at improving perfor- motolerant, fast-growing strain of C. sorokiniana, and
mances in PBRs. selected pale-green mutants by imaging Chl fluorescence.
Mutants were able to perform photosynthesis more effi-
Promises of domestication by forward genetic ciently than wild type, minimizing photoinhibition in
in improving photosynthetic efficiency high light; the positive effect on photosynthetic produc-
According to the previous section, a gap between theo- tivity was confirmed in both lab-scale and outdoor PBRs.
retical and real biomass productivities of microalgae Similar results were obtained with N. gaditana strains
originates from the high OD of cells. With respect to having reduced cellular pigment content [102]. Finally,
the problem of inhomogeneous light distribution, high simultaneous knock-down of three light-harvesting com-
density planting of crops is a condition limiting PAR plex proteins (LHCMB1, 2 and 3) in C. reinhardtii, by an
penetration thus productivity, and it is not different RNAi triple knock-down strategy, resulted in improved
than the condition of elevated cell density reachable in a light-to-H2 (+ 180% than wild type) and light-to-biomass
PBR. Moreover, biomass production with wild type algal (+ 165%) conversion efficiencies [103].
strains is poorly viable likely as farming with ancestral Implementation of biosynthetic pathway of accessory
crop varieties. pigments, e.g. phycobilins or Chls, into genus of indus-
The so called ‘Green Revolution’ of agriculture, a trial interest, has been proposed for improving harvest-
domestication based on breeding and phenotypic selec- ing efficiency over the full PAR spectrum [4]. Recently,
tion, succeeded in pursuing crop productivities over the the enzyme responsible for the synthesis of Chl f, an oxi-
past 50 years [92, 93]. Industrial application of microal- dized form of Chl a, has been isolated from the cyano-
gae may take advantage of a domestication approach, bacterium C. fritschii [104]; heterologous expression of
analogous to that carried out for modern crops. Thus, Chl f synthase succeeded in accumulating this chromo-
selection of strains carrying desired traits, together with phore in Synechococcus sp. Since Chl f expands the spec-
implementing new alleles by random mutagenesis or tral range for photosynthesis by absorbing far red light,
genetic engineering, might improve performances in its expression in microalgae may confer advantages
PBRs. for mass culture in PBRs, which suffers for detrimental
Random mutagenesis is recognized as a powerful tech- sieve-effects at high cell densities. However, the feasibil-
nology in mutation breeding, widely employed for strain ity of this approach, which assumes a correct binding of
improvement and for studying the molecular basis of the new chromophores into the existing pigment-binding
metabolic processes. Forward genetic approach is of par- complexes, still await experimental confirmation.
ticular relevance for algal biotechnology, since it avoids CO2 fixation rate is a major limiting step in biomass
restrictions to GMO for outdoor production system [94]. yield, which arise from RuBisCO inefficiency. Genetic
The most common method for generating genetic vari- engineering of RuBisCO to increase its catalytic activ-
ability in a population of microalgae is the mutagenesis ity or to enhance its specificity towards CO2 have been
induced by either physical methods (UV-light, γ- and proposed as ways to overcome these limitations [4]. It
X-rays) or chemical mutagens, e.g. N′-nitro-N nitroso- is worth noting that the natural diversity of RuBisCO
guanidine (NTG) and ethyl methanesulfonate (EMS). is limited, likely because the interactions which sup-
Attempts for algae genetic improvement, aimed to port catalytic activity make most of the isoforms of this
enhance light-to-biomass conversion efficiency, relied on enzyme intolerant to mutations [90], indeed attempts to
random mutagenesis and screening of favorable traits. overcome its limitations by directed evolution, had scant
These approaches, while overcame scarcity of genetic success. Although variants of RuBisCO with higher activ-
engineering tools in microalgae, needed for efficient ity have been identified [105, 106] their heterologous
screening strategies for strains with higher productivity. expression in algal strains of industrial potential is still
Some of these approaches, which succeeded in increasing missing. Recently, E. coli-based screen of new RuBisCO
photosynthetic yield, are presented as follows. variants obtained by direct evolution allowed the identi-
Due to detrimental effect of high OD for mass cul- fication of an unexplored subunit interface with poten-
tivation, strains carrying truncated antenna size were tial of increasing CO2 fixation rate [107]. Site-directed
mutagenesis in such subunit may generate novel variants Microalgae growing in mass culture experience rapid
whose enzymatic characteristics can be subsequently changes in the irradiance due to cell mixing into the
tested in microalgae in terms of enhanced CO2 fixation. PBR. The amount of time spent in sub-saturating vs.
An additional approach may reside in the generation of excess light influences the biomass productivity, which
hybrid RuBisCO by using novel activase isoforms from is lower in fluctuating light conditions [115] possibly due
chemolithoautotroph microorganisms such as Acidithio- to the metabolic energy needed to repair photodamage.
bacillus ferrooxidans [108]. Hence, improving photosynthetic efficiency in excess
A high photosynthetic efficiency is attainable only light conditions is potentially a major goal for establish-
in low irradiance and controlled light environments, ing efficient outdoor cultivation. Research efforts aimed
which allow most absorbed photons can be utilized by to obtain non-GMO algal strain tolerant to excess light,
the culture; instead in the outdoor, efficiency drops due mainly focused on the model alga C. reinhardtii. Förster
to fluctuating irradiances, exceeding the photosynthetic et al. [117] isolated very high light resistant (VHL-R)
capacity. Autotrophs evolved mechanisms for regulating mutations, which allowed near maximal growth rate at
the efficiency of light capture, which can become target irradiances lethal to the control genotype; characteriza-
of domestication strategies. Several microalgal species tion of these strains reveals they affected the regulatory
e.g. C. reinhardtii and H. pluvialis, trigger fast phototac- pathways which modulate photoprotective response,
tic response, for fine-tuning exposure to light. Indeed, including PSII repair and ROS detoxification. In [118],
phototaxis confers fitness advantage and it is regulated wild type strain was UV-mutagenized and plated onto
by cytoplasmic redox balance, which in turn is affected medium containing a lethal concentration of the photo-
by photosynthetic electron transport rate [109]. In the sensitizer Red Bengal; by this approach, SOR1 was iden-
attempt of isolating strains with improved light-use-effi- tified as a factor enhancing tolerance to photooxidative
ciency, Kim and collaborators [110] analyzed a C. rein- stress conditions. Schierenbeck and coworkers [119]
hardtii mutant population for rapid phototaxis response performed UV-mutagenesis followed by selection under
and identified mutants with enhanced photoautotrophic high irradiance (2000 μmol m−2 s−1); the two muta-
growth and lipid production, respectively 1.9- and 8.1- tions selected, which both mapped in the putative Light
fold increases than wild type. Responsive Signal 1 (LSR1) gene, conferred an improved
Photosynthetic organisms dynamically regulate the resistance of cells against exogenous ROS. Recent results
amplitude of NPQ (see “Biological constraints in light- concern the isolation of pale-green, singlet oxygen resist-
to-biomass conversion efficiency” section): by balanc- ant mutant by EMS-mutagenized C. vulgaris, which
ing amplitudes of light harvesting vs. energy dissipation, showed biomass yield enhancement by 68% than wild
they maintain optimal fitness in changing light environ- type strain (Dall’Osto et al. unpublished results).
ment. The slow relaxation rate of NPQ upon high- to
low-light transition was considered to reduce the over- Improving algal biomass productivity by genetic
all conversion efficiency of solar to biomass in micro- engineering: methods, state of the art and perspectives
algae, consistent with recent evidences in plants [111, Genetic manipulation approaches have the potential
112]. Indeed, deletion of the OCP protein, responsi- to revolutionize industry based on microalgae cultiva-
ble for NPQ response in cyanobacteria, resulted in a tion. These include transfer of genes isolated from other
30% higher biomass yield in mass cultures of Synecho- species to generate strains with desirable commercial
cystis than wild type cells [113]. Random insertional traits such as tolerance to excess light and heat stress,
mutagenesis and Targeting Induced Local Lesions IN resistance to herbivore/pathogen, capacity to outcom-
Genomes (TILLING) approach on C. reinhardtii, follow- pete opportunistic organisms, or to express biosyn-
ing by Chl fluorescence imaging screening, has produced thetic pathways into more productive strains (Fig. 3).
mutants specifically devoid of lhcsr genes [87, 114]. In Recent progress in genome sequencing, methagenome/
their report, [115] proposed that biomass productivity metatranscriptome approaches, and genetic manipula-
depends on LHCSR protein accumulation: C. reinhardtii tion, yielded into significant advancement in microalgal
strains lacking the two lhcsr3 genes were more produc- research. In this paragraph, different strategies of genetic
tive than wild type, thus confirming down-regulation of engineering, which revealed effective in improving algal
NPQ is a strategy for improving light use efficiency in productivity, are discussed. Moreover, additional solu-
microalgae. Instead, more recently, [116] observed no tions are proposed.
significant differences in biomass yield between C. rein- In the last decades, several efforts have been attempted
hardtii wild type and the npq4 lhcsr1 mutant, devoid of to optimize the transformation efficiency of differ-
all Lhcsr isoforms. ent microalgae species. Stable transformation was first
developed in C. reinhardtii: being able to growth both in
Fig. 3 Schematic depiction of the major desirable traits to be either implemented or improved, toward higher productivity of microalgae in mass
culture
autotrophic and heterotrophic conditions, as haploid or by fusing the sequences encoding the gene of interest and
diploid cells, Chlamydomonas was adopted as powerful the selection marker in a unique bicistronic RNA [132,
genetic system for studying different physiological mech- 133]. Recourse to a frequent subset of preferred codons
anisms. Nuclear transformation of C. reinhardtii may results in elevated transcriptional levels, while the use
be achieved by several methods such as electroporation of codons introducing unintended splicing signals nega-
[120], Agrobacterium-mediated transformation [121], tively affects the overall expression of transgenes [134],
silicon carbide whiskers and positively-charged amino- thus a codon usage optimization is mandatory to maxi-
clay nanoparticles [122, 123] and glass beads agitation mize protein yield. To improve the selection of high-level
method [124]. Both electroporation and Agrobacterium- expressing transformants, last-generation expression vec-
mediated transformation also succeeded in transforming tors exploit the 2A peptide [135] to generate transcrip-
algae of economic interest such as C. vulgaris, Neochloris tional fusions between selection marker sequence (e.g.
oleoabundans and H. pluvialis [125–127]. The introduc- antibiotic resistance) and the gene of interest [132]. Tran-
tion of foreign genes into the nuclear genome of micro- scription factors are gaining increasing attention as key
algae is generally guided by a random integration event regulators of metabolic pathways, in order to enhance
[128]. Nuclear gene expression is frequently subjected to yield of high-value molecules or to maximize the produc-
strong silencing mechanisms due both to position effect tion of foreign proteins in microalgae. Over-expression of
and to epigenetic phenomena, similar to those of land NsbZIP1, a transcription factor carrying the basic leucine
plants [129]; indeed in microalgae, silenced multicopy zipper, resulted in both enhanced growth rate and higher
transgenes exhibit high levels of DNA methylation as in lipid contents in N. salina [136].
land plants [130, 131]. The biolistic method (particle-gun bombardment) is
In last years, many efforts have been attempted to the elective procedure for chloroplast transformation
increase the heterologous expression potential of micro- in microalgae [137]. The introduction of foreign genes
algae. Increased expression of transgenes was obtained into the plastome is guided by site-specific integration
event (i.e. by homologous recombination). Expressing can be efficiently performed by introducing expression
foreign genes in the chloroplast enables to circumvent cassettes obtained by PCR, making unnecessary the use
gene silencing events, which affect the nuclear expres- of expression vectors [144]. Recently, a doubling of the
sion; moreover, it allows for the introduction of operons, lipid production in N. gaditana was obtained by deleting
encoding several enzymes of a pathway. However, the a transcription factor that acts as negative regulator in
resulting transformants must undergo several rounds of lipid biosynthesis [145].
selection in order to acquire the homoplasmic condition As previously described, a truncated antenna size
[138]. yielded into increased productivity in green microal-
Further details concerning nuclear and chloroplast gae (see “Promises of domestication by forward genetic
expression in C. reinhardtii are summarized in Table 2; in improving photosynthetic efficiency” section), thus
moreover, many of these aspects have been covered proteins involved in the biogenesis of photosynthetic
extensively in a recent review [137]. machinery can be targeted for increasing biomass pro-
Genome editing technology enables for both gene dele- duction. Truncated light-harvesting antenna 1 (TLA1),
tion and gene integration, therefore implementation of a nuclear gene putatively involved in the regulation of
these novel genetic tools in microalgae would allow for the antenna size of C. reinhardtii, was up- and down-
manipulation of metabolic networks. Recently, a novel regulated by overexpression and RNAi, respectively. The
approach based on CRISPR–CAS9 genome editing tech- strain over-expressing TLA1 showed a larger antenna
nology have been successfully developed in both the size for both photosystems and lower Chl a/b ration than
marine diatom P. tricornutum [139] and in C. reinhardtii, the wild type, while its down-regulation resulted in the
allowing for deletion of specific gene functions [140]; the opposite phenotype changes [146]. LHCII, the nucleus-
latter was achieved by a DNA-free CRISPR–Cas9 method encoded light-harvesting proteins associated with PSII,
and the outcome was the sequential FTSY and ZEP dou- tunes the light harvesting capacity to the prevailing light
ble-gene knockout, that resulted in improved photosyn- condition. In C. reinhardtii, LHCII translation efficiency
thetic productivity. Analogous approach was used to is regulated by the cytosolic RNA-binding protein NAB1,
abolish the functions encoded by MAA7, CpSRP43 and which is subjected to specific nitrosylation in limiting
ChlM genes, which led to pale-green mutants [141]. In light, thus making such repressor less-active and promot-
C. reinhardtii, the replacement of Cas9 with the Cpf1 ing accumulation of LHC [147].
ribonucleoprotein achieved a more efficient homology- Manipulation of RuBisCO activity, namely the major
directed DNA replacement [142]. However, a common constraint for C assimilation e.g. under excess light
limitation of free-DNA CRISPR–Cas9/Cpf1 methods conditions [148], may improve the photosynthetic
resides in the lack of selection markers that, in turn, hin- yield [149]. Although site-directed mutants in the rbcL
ders a straightforward selection of the desired mutants; (RuBisCO large subunit) gene [150, 151] as well as hybrid
being the genome editing event induced at very low fre- variants with altered specificity of RuBisCO–RuBisCO
quency (0.5–0.6%), a visible phenotype makes mutant activase interaction [152] have been generated, their
selection easier. A DNA-based CRISPR–CAS9 method over-expression in C. reinhardtii did not increase the
has been developed in the industrial oleaginous micro- overall photosynthetic yield.
alga N. oceanica [143] in which nuclear transformation
Table 2 Transformation of C. reinhardtii

Organelle DNA-delivery method Genetic mechanism Advantages Disadvantages Selection marker
Nucleus Electroporation Ectopic recombination Protein can be expressed Identification of Resistance to Paromycin,
gene-gun bombarment (random integration) as secreted protein high-expressing Zeocin, Hygromycin, Chloram-
A. tumefaciens-mediated Post-translational modi- Transformants phenicol
Glass beads fications gene silencing Auxotrophic complementation
Silicon carbide whiskers and (ARG7, NIT1, oee1)
aminoclay nanoparticles
Chloroplast Gene-gun bombarment Homologous recombi- Compartmentalization Need to identify resistance to Spectinomycin
Glass beads nation (site-specific Lacks gene silencing homoplasmic Auxotrophic complementation
integration) High expression level transformant (atpB, psbH)
Lacks post-
translational
modifications
This table displays DNA-delivery methods, genetic mechanism driving the transformation, and the most common selection markers employed so far. Major
advantages and disadvantages of nuclear vs. chloroplast transformation are reported
Recently, biomass productivity as well as lipid yield Other algal species can metabolize a large array of
increased up to 40% in the oleaginous Nannochlorop- sugars, and strongly increase their productivity under
sis oceanica by overexpressing endogenous RuBisCO heterotrophic or mixotrophic growth conditions [162];
activase [153]. Conversely, a reduction in the RuBisCO however, heterotrophic growth requires additional costs
activity by site directed mutagenesis resulted in a ten- due to need for exogenous carbon source and mainte-
fold higher H 2 production in C. reinhardtii [154], likely nance of axenic conditions. Algal strains able of metabo-
because Calvin-Benson cycle competes with Hydroge- lizing raw lignocellulosic biomass scraps, namely cheap
nase for reducing equivalents. agricultural wastes, would certainly contribute to make
Further strategies included (i) the PCR-based gene the whole process economically viable. Foreign genes
shuffling of Chlamydomonas rbcL with sequences repre- encoding bacterial and fungal plant Cell Wall Degrad-
senting natural variants of this gene, which yielded iso- ing Enzymes (CWDEs), were constitutively expressed in
forms with higher Vmax of carboxylation catalysis [155]; microalgae and addressed to the secretory pathway [132].
(ii) regulation of RuBisCO accumulation according to In C. reinhardtii, yield of secreted proteins was improved
culture conditions, by tuning mRNA level of the nuclear up to eight-fold by fusing both the putative signal pep-
maturation factor MRL1 [156]; (iii) overexpression of. tide of gametolysin and the repeated serine-proline mod-
sedoheptulose 1,7-bisphosphatase from C. reinhardtii, ule, to the N and C terminus of the recombinant protein,
which succeeded in enhancing photosynthetic efficiency respectively [163]. Contrary to plant cell, some species of
in D. bardawil [157]; finally, the over-expression of Low- unicellular green algae possess a cell wall mainly consti-
CO2 Inducible (LCI) proteins in C. reinhardtii, under tuted by proteins (e.g. C. reinhardtii) [164], thus lack of
conditions which typically repress their synthesis (i.e. polysaccharides as major components circumvents the
high CO2 concentration), increased biomass production deleterious effects of expressing CWDEs in plants, likely
under elevated C O2 conditions as much as 80% than con- related to hyper-immune responses [165, 166]. Although
trol strain [158]. some algal spp. synthesizes endogenous CWDEs [167],
High productivity in open ponds is restricted to species the native cellulolytic machinery is not efficient enough
which adapted to high salt concentration (e.g. Dunaliella) for degrading hydrolysis-recalcitrant substrates such
or high pH (e.g. Spirulina), thus outcompeting naturally as lignocellulose. Thus, a promising perspective is the
occurring contaminants. Hence, a trait which confers expression of a range of secreted CWDEs, includ-
competitive advantage over undesirable microorganisms, ing polygalacturonases, hemicellulases, cellulases and
is crucial both to increase the biomass productivity and ligninases in a unique algal culture, analogously to the
to reduce the operating costs for maintenance of axenic approaches developed in yeasts which yielded into strains
cultures (Fig. 1), particularly in either open pond or het- able to grow on cellulosic substrates [168].
erotrophic conditions. In this perspective, non-canonical An overview of the major genetic manipulations which
substrates may be employed for sustaining algal growth. may lead to an improvement of biomass productivity is
The expression of the phosphite dehydrogenase D represented in Fig. 4.
(PTXD) from P. stutzeri WM88 [159] confers to C. rein-
hardtii the capacity of metabolizing phosphite, namely Conclusions
a P source which cannot be utilized by plants, fungi and Commercially cultivated for several decades, micro-
most bacteria. Transgenic Chlamydomonas cells showed algae are now recognized to offer a great potential for
higher fitness than S. obliquus in competition experi- exploitation in different fields including pharmaceu-
ments in which phosphite-repleted/phosphate-depleted ticals, aquaculture and renewable energies. Former
medium was used [160]. efforts in their industrial applications mainly focused
Some algal species are strict autotrophs or are highly in optimizing culture parameters and selecting the
selective for their C source (e.g. Chlamydomonas for best performing wild type strain. However, to promote
acetate), thus trophic conversion by metabolic engineer- cultivation of microalgae as a new biotechnological
ing would be desirable. Chlamydomonas cells expressing sector, a number of challenges still have to be over-
the hexose transporter HUP1 (monosaccharide-H+ sym- come. Domestication strategies achieved by genetic
porter from C. kessleri) metabolized externally supplied and metabolic engineering will be crucially impor-
glucose for heterotrophic growth and showed higher H2 tant to isolate “smart strains” with improved yield, in
production capacity; however, results suggest that glu- order to make the production successfully marketed.
cose cannot fully replace acetate as a C source, for long- The opportunities offered by investments in both basic
term growth in the dark [161]. and applied research, are considerable: (i) the rapid
evolution of genome sequencing techniques will help
Fig. 4 Potential traits to be implemented in GM—C. reinhardtii cell. The diagram displays a number of genetic strategies, aimed to enhance
productivity in mass culture of microalgae. Gene over-expression (OE) using hydrid promoters or viral cis-acting elements and gene disruption/
down-regulation (KO/KD) by Crispr–Cas9 and RNAi approaches are indicated. Some traits that may result in higher productivity include an increased
photosynthetic efficiency, improved phototaxis, the use of non-canonical substrates, and optimized carotenoid, lipid and isoprene metabolism.
Up- and down-ward pointing arrow mean up- and down-regulation, respectively, and are referred to the expression level of the corresponding
endogenous enzyme. Bulb and red cross mean enzymatic in vitro improvement and loss of function, respectively. Abbreviations: Chl-f S chlorophyll
f synthase, CWDE cell-wall degrading enzyme, FTSY chloroplast signal recognition particle, GL gametolysin signal peptide, HS hydrocarbon-synthase,
HUP1 hexose-proton symporter, LHC light harvesting complexes, ME malate dehydrogenase, ML multifunctional lipase, NAB1 RNA-binding protein,
PHY D phytoene desaturase, PHY S phytoene synthase, PS patchoulol synthase, PTXD phosphite dehydrogenase, β-PS β-phellandrene synthase, TF
transcription factor, TLA1 truncated light-harvesting antenna 1, ZEP zeaxanthin epoxidase
defining the gene networks controlling growth, while of the primary metabolism to the mass culture condi-
-omics approaches allow to identify regulatory points tions, or the development of molecular strategies for
of cellular pathways, thus enabling manipulation of key strain containment. Encouraging results have recently
metabolic steps; (ii) prospective redesigns of algal sys- been obtained by boosting light-use-efficiency or by
tem include light-to-biomass conversion efficiency, oil strengthening specific metabolic pathways. Additional
content/composition, nutrient recovery capacity; (iii) research efforts and funding for implementing inno-
extend the genetic transformation techniques, now car- vative biorefineries, will realistically support progress
ried out successfully in few species only, to the most toward next-generation algal biotechnology.
industrially-relevant species, will offer the opportunity
to address the biological constraints limiting growth
Abbreviations
yield; finally, (iv) the development of reproducible Chl: chlorophylls; CWDE: cell wall degrading enzyme; DHA: docosahex-
genome editing techniques will permit a fine matching aenoic acid; DW: dry weight; EMS: ethyl methanesulfonate; EPA: eicosap-
entaenoic acid; GMO: genetically-modified organism; lc-PUFA: long-chain
polyunsaturated fatty acid; LHCBM/LHCSR: light-harvesting complex proteins; 11. Scranton MA, Ostrand JT, Fields FJ, Mayfield SP. Chlamydomonas
NGT: N′-nitro-N nitrosoguanidine; NPQ: non-photochemical quenching; OD: as a model for biofuels and bio-products production. Plant J.
optical density; PAR: photosynthetically active radiation; PBR: photobioreactor; 2015;82:523–31.
PSII: photosystem II; ROS: reactive oxygen species; RuBisCO: ribulose 1,5-bis- 12. Sutherland DL, Howard-Williams C, Turnbull MH, Broady PA, Craggs RJ.
phosphate carboxylase-oxygenase; RuBP: ribulose 1,5-bisphosphate; TAGs: Enhancing microalgal photosynthesis and productivity in wastewater
triglycerides; TW-y: terawatts-year. treatment high rate algal ponds for biofuel production. Bioresour
Technol. 2015;184:222–9.
Authors’ contributions 13. Dineshkumar R, Kumaravel R, Gopalsamy J, Sikder MNA, Sampathkumar
MB and LD identified patterns and trends in the literature and designed the P. Microalgae as bio-fertilizers for rice growth and seed yield productiv-
structure of the review. VV and SB prepared the tables and figures. All of the ity. Waste Biomass Valoriz. 2018;9:793–800.
authors contributed to searching for relevant literature, and carried out a criti- 14. Passos F, Uggetti E, Carrère H, Ferrer I. Pretreatment of microal-
cal analysis of the literature, discussed together and wrote the manuscript. All gae to improve biogas production: a review. Bioresour Technol.
authors read and approved the final manuscript. 2014;172:403–12.
15. Garcia-Gonzalez J, Sommerfeld M. Biofertilizer and biostimulant
properties of the microalga Acutodesmus dimorphus. J Appl Phycol.
Acknowledgements 2016;28:1051–61.
We thank Prof. Roberto Bassi for his suggestions made during the preparation 16. Bhola V, Swalaha F, Ranjith Kumar R, Singh M, Bux F. Overview of
of this review. Because of space constraints, a number of noteworthy publica- the potential of microalgae for C O2 sequestration. Int J Environ Sci
tions has not been cited or discussed properly. We apologize to the authors Technol. 2014;11:2103–18.
for this lack of completeness. 17. Zhu X-G, Long SP, Ort DR. improving photosynthetic efficiency for
greater yield. Annu Rev Plant Biol. 2010;61:235–61.
Competing interests 18. Melis A. Solar energy conversion efficiencies in photosynthesis:
The authors declare that they have no competing interests. minimizing the chlorophyll antennae to maximize efficiency. Plant
Sci. 2009;177:272–80.
Availability of data and materials 19. Rodolfi L, Zittelli GC, Bassi N, Padovani G, Biondi N, Bonini G, et al.
Not applicable. Microalgae for oil: strain selection, induction of lipid synthesis and
outdoor mass cultivation in a low-cost photobioreactor. Biotechnol
Consent for publication Bioeng. 2009;102:100–12.
Not applicable. 20. Chisti Y. Biodiesel from microalgae. Biotechnol Adv. 2007;25:294–306.
21. Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel produc-
Ethics approval and consent to participate tion and other applications: a review. Renew Sustain Energy Rev.
Not applicable. 2010;14:217–32.
22. Mitra D, van Leeuwen (Hans) J, Lamsal B. Heterotrophic/mixotrophic
Funding cultivation of oleaginous Chlorella vulgaris on industrial co-products.
Work in our lab is supported by grants from the University of Verona (Fondo Algal Res. 2012;1:40–8.
Ricerca di Base HuntingLight—Grant Number 201190). 23. Cuaresma M, Janssen M, Vílchez C, Wijffels RH. Productivity of Chlo-
rella sorokiniana in a short light-path (SLP) panel photobioreactor
under high irradiance. Biotechnol Bioeng. 2009;104:352–9.
Publisher’s Note 24. Herrera Bravo de Laguna I, Toledo Marante FJ, Luna-Freire KR, Mioso
Springer Nature remains neutral with regard to jurisdictional claims in pub- R. Extraction of nutraceuticals from Spirulina (blue-green alga): a
lished maps and institutional affiliations. bioorganic chemistry practice using thin-layer chromatography.
Biochem Mol Biol Educ. 2015;43:366–9.
Received: 8 July 2018 Accepted: 31 October 2018 25. Al-Dhabi NA, Valan Arasu M. Quantification of phytochemicals from
commercial spirulina products and their antioxidant activities. Evid
Based Complement Alternat Med; 2016. Article ID: 7631864.
26. Hynstova V, Sterbova D, Klejdus B, Hedbavny J, Huska D, Adam V.
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Biomass From Microalgae: The Potential of Domestication Towards Sustainable Biofactories

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Benedetti et al.

Microb Cell Fact (2018) 17:173

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Biomass from microalgae: the potential

Background storing it efficiently. Oxygenic photosynthesis converts

Table 2 Transformation of C. reinhardtii

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