The right stuff; realizing the potential for enhanced biomass production in microalgae

Abstract

There is growing evidence that eukaryotic microalgae can become a more sustainable and profitable alternative than terrestrial crops to produce feed, fuels, and valuable coproducts. The major factor driving progress in algal biomass production is the potential of microalgae to produce substantially greater biomass per unit land area than terrestrial crops. To be financially feasible, however, current algal biomass yields must be increased. Given the fact that algal biomass production is in its infancy there exist multiple opportunities to improve biomass yields. For example, recent bioprospecting efforts have led to the identification of new microalgal strains having biomass yields that compete economically with plant biomass. Substantial increases in biomass yields have also been achieved using advanced genetic engineering approaches. Targeted improvements in photosynthetic efficiency have led to three-fold increases in algal biomass yields. One genetic tool that has seen limited application for algal biomass enhancement is advanced breeding genetics. The greater availability of algal genomes and recent advancements in breeding algae will further accelerate yield improvements. Genetic engineering strategies to increase biomass production will also be assisted by transcriptomic and metabolomic studies that help identify metabolic constraints that limit biomass production. In this review we assess some of the recent advances in algal strain selection, directed evolution, genetic engineering and molecular-assisted breeding that offer the potential for increased algal biomass production.

Full text

The right stuff; realizing the
potential for enhanced biomass
production in microalgae
Sowmya Subramanian
1
and Richard T Sayre
2
*
1
Thermo Fisher Scientific, Alachua, FL, United States,
2
New Mexico Consortium, Los Alamos, NM,
United States
There is growing evidence that eukaryotic microalgae can become a more
sustainable and profitable alternative than terrestrial crops to produce feed,
fuels, and valuable coproducts. The major factor driving progress in algal
biomass production is the potential of microalgae to produce substantially
greater biomass per unit land area than terrestrial crops. To be financially
feasible, however, current algal biomass yields must be increased. Given the
fact that algal biomass production is in its infancy there exist multiple
opportunities to improve biomass yields. For example, recent bioprospecting
efforts have led to the identification of new microalgal strains having biomass
yields that compete economically with plant biomass. Substantial increases in
biomass yields have also been achieved using advanced genetic engineering
approaches. Targeted improvements in photosynthetic efficiency have led to
three-fold increases in algal biomass yields. One genetic tool that has seen
limited application for algal biomass enhancement is advanced breeding
genetics. The greater availability of algal genomes and recent advancements
in breeding algae will further accelerate yield improvements. Genetic
engineering strategies to increase biomass production will also be assisted
by transcriptomic and metabolomic studies that help identify metabolic
constraints that limit biomass production. In this review we assess some of
the recent advances in algal strain selection, directed evolution, genetic
engineering and molecular-assisted breeding that offer the potential for
increased algal biomass production.
KEYWORDS
microalgae, biomass, biofuels, photosynthesis, genetics, carbon capture
Highlights
•Based on thermodynamic considerations five-fold increases in algal biomass
production are theoretically feasible
OPEN ACCESS
EDITED BY
Blake Hovde,
Biosciences Division, Los Alamos
National Laboratory (DOE),
United States
REVIEWED BY
Weiqi Fu,
Zhejiang University, China
Tonmoy Ghosh,
Indian Institute of Technology Indore,
India
*CORRESPONDENCE
Richard T Sayre,
richardtsayre@gmail.com
SPECIALTY SECTION
This article was submitted to Bioenergy
and Biofuels,
a section of the journal
Frontiers in Energy Research
RECEIVED 27 June 2022
ACCEPTED 23 August 2022
PUBLISHED 13 September 2022
CITATION
Subramanian S and Sayre RT (2022), The
right stuff; realizing the potential for
enhanced biomass production
in microalgae.
Front. Energy Res. 10:979747.
doi: 10.3389/fenrg.2022.979747
COPYRIGHT
© 2022 Subramanian and Sayre. This is
an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY).
The use, distribution or reproduction in
other forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which does
not comply with these terms.
Abbreviations: Bu, bushels; CBBC, Calvin-Benson-Bassham Cycle; Chl, chlorophyll; gdw, gram dry
weight.
Frontiers in Energy Research frontiersin.org01
TYPE Review
PUBLISHED 13 September 2022
DOI 10.3389/fenrg.2022.979747

•Enhanced and stable algal biomass production strains have
recently been identified that achieve biomass yields
approaching those necessary for economical bioproduct
production
•Molecular assisted breeding of algae has been shown to
enhance biomass yields more than three fold
•Engineering the photosynthetic apparatus of algae has lead
to three-fold increases in biomass production
•Future strategies for enhancing biomass production in
algae are considered
Introduction
Among the major constraints limiting the commercialization
of eukaryotic microalgae for biomass production are the high
costs of cultivation, harvesting and processing. Relative to crop
plants algal biomass production requires substantial capital and
operating investments including lined ponds, pond aeration and
mixing systems, and energy intensive algal harvesting and
processing systems (Unkefer et al., 2017;Benedetti et al., 2018;
Sun et al., 2018;Rajvanshi and Sayre, 2020). The high operating
costs for algal biomass can largely be attributed to the fact that
algal biomass concentration is only 0.1%–0.5% of the cultivation
media substantial volumes of water must be managed to grow
and harvest algae requiring specialized infrastructure and high
energy costs that could exceed the energy content of the algal
biomass. Offsetting these extensive cost factors, however, is the
high growth rate of algae. High algal growth rates are associated
with their potential to grow throughout the year, the absence of
heterotrophic tissues not engaged in biomass production, and the
presence of high efficiency photosynthetic systems including
active CO
2
concentrating systems that reduce photorespiratory
carbon losses (Sayre, 2010;Wang et al., 2011;Subramanian et al.,
2013). As a result, algae have the potential to produce as much as
5-fold more biomass per unit land area than terrestrial crop
plants (Weyer et al., 2010;Subramanian et al., 2013).
Consequently, there has been incredible interest to develop
microalgae for low-cost production of feeds, fuels, high value
bioproducts and to sequester atmospheric carbon dioxide to
mitigate climate change (Olivares et al., 2016;Lammers et al.,
2017;Unkefer et al., 2017;Marrone et al., 2018;Sayre, 2010).
However, there remain multiple constraints that limit the
commercialization of eukaryotic microalgae. Life cycle and
techno-economic analyses of algal cultivation systems have
indicated that only open-pond production systems can
economically produce algal biomass that is cost competitive
with crops (Chisti, 2013;Davis et al., 2014;Barry et al., 2016).
But to be economically feasible, outdoor open-pond cultivation
systems must produce stable biomass yields approaching 40 gdw/
m
2
/day, corresponding to an overall solar to biomass conversion
efficiency of ~3% (Bolton and Hall, 1991;Kruse et al., 2005a;
Kruse et al., 2005b;Zhu et al., 2008;Lammers et al., 2017). Given
that the maximum theoretical efficiency for solar energy
conversion into the chemical energy of algal biomass by
photosynthesis is approximately 11% there is the potential to
achieve 2- to 3-fold increases in biomass yields (Bolton and Hall,
1991;Kruse et al., 2005a;Kruse et al., 2005b;Zhu et al., 2008;
Weyer et al., 2010). Thus, substantial improvements in biomass
accumulation are theoretically possible.
When considering strategies to increase algae biomass yields
it is informative to consider genetic and agronomic practices that
have been successfully used to increase biomass production in
terrestrial crops. Over the last 75 years great strides have been
GRAPHICAL ABSTRACT
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achieved in the development of higher yielding and more robust
crops. These achievements have largely been the result of
advanced agronomic practices to better manage resource
inputs and the application of modern agricultural breeding
techniques to improve crop genetics. Crop breeding strategies
were initially based on the application of genetic linkage analyses
between traits of interest and more tractable or scorable traits. In
the last 40 years molecular assisted breeding and genomic
selection techniques have further accelerated the development
of improved crops. The application of modern breeding systems
including, the use of inbred lines, hybrid sterility mechanisms,
and transgenic modifications, has led to over a 7-fold increase in
maize yields between 1940 and 2015 (168 bu/acre). Most of these
improvements in crop yield have been achieved through the
development of herbicide resistance genes, and the introduction
of pest, pathogen and stress tolerance traits. Surprisingly, few
projects focusing enhanced carbon capture for biomass yield
improvements have reached the stage of commercial
deployment.
In contrast to terrestrial crops, the greatest enhancements in
microalgal biomass yields have been realized through the
identification of more robust algal species and through
directed selection and transgenic approaches rather than
through traditional breeding approaches (Kumar et al., 2013;
Kumar et al., 2016;Crozet et al., 2018;Fayyaz et al., 2020;
Dementyeva et al., 2021;Mosey et al., 2021). In the following
sections we address why that is the case and review recent
advances that have led to further improvements in eukaryotic
microalgal biomass production.
Strain selection for enhanced biomass
yield
The emerging consensus is that the most economically
feasible means to produce algal biomass is to cultivate algae in
continuously mixed, open pond culture systems (Chisti, 2013;
Davis et al., 2014;Barry et al., 2016;Lammers et al., 2017;
Marrone et al., 2018). These pond systems typically range in
depth from 15 to 30 cm. While greater algal biomass yields per
unit area are possible in closed photobioreactor (PBR) systems
these systems have several inherent disadvantages when operated
at large scale including, greater capital and operating expenses
than open pond systems, challenges associated with the
formation of biofilms that reduce light penetration and the
management of pathogens or herbivores (Chisti, 2013;Davis
et al., 2014;Barry et al., 2016;Lammers et al., 2017;Marrone
et al., 2018;Rajvanshi and Sayre, 2020). Overall PBR-based algal
biomass production systems are estimated to be 2- to 2.5-fold
more expensive to operate than open pond production systems
(Chisti, 2013;Davis et al., 2014;Barry et al., 2016). Due to their
open nature algal species that are cultivated in ponds must have
fast growth rates to out compete weedy invasive species and be
resistant to biotic and abiotic stressors including large
temperature and light intensity shifts, limiting daylength
periods at high latitudes, precipitation, competing weedy algal
species, pathogens, and herbivores (Barry et al., 2016;Neofotis
et al., 2016;Olivares et al., 2016;Marrone et al., 2018;Rajvanshi
and Sayre, 2020). As a result of these challenges substantial efforts
have gone into screening or bioprospecting for algal strains that
not only have high growth rates but that can also tolerate abiotic
or biological stressors that lead to pond crashes. Given that there
are over 164,000 known species of algae bioprospecting for high
biomass producing algal strains particularly those best suited for
the local environments where they are cultivated continues to
provide opportunities for enhancing algal biomass yields
(Marrone et al., 2018;Guiry and Guiry, 2021).
Since the mid-1970s, the United States Department of Energy
has supported multiple algae bioprospecting efforts to identify
high biomass yielding and stable strains suitable for commercial
biomass production (Weissman and Goebel, 1987;Barry et al.,
2016;Olivares et al., 2016). To date, algal strains having the
highest and most stable biomass production rates are dominated
by species from two phyla, the Chlorophyta and Ochrophyta.
These include species from the genera, Chlorella, Picochlorum,
Scenedesmus, Cyclotela, Tetraselmus and Nanochloropsis
(Table 1). Many of these species have demonstrated long-term
biomass production (>100 days) yields approaching 40 gdw/m
2
/
day 22, (Huesemann et al., 2018;Dahlin et al., 2019;Gonzalez-
Esquer et al., 2019;Aketo et al., 2020;Mucko et al., 2020;Cano
et al., 2021;Krishnan et al., 2021). Averaged over a yearlong
growing season algae having a growth rate of 40 gdw/m
2
/day
could yield as much as 145 metric tons dw/ha/yr having an
average energy density of 21 kJ/gdw. In comparison, corn yields
in the United States Midwest average 11.5 metric tons dw/ha/yr
having an average energy density of 16.7 kJ/gdw. Thus, the
annual areal energy yield for algal biomass grown at a rate of
40 gdw/m
2
/day is 16-fold greater than for corn. Given that the
corn growing season is only 3 months, however, the relative
efficiency of algal biomass production is about 4-fold greater than
for corn on an annualized basis.
Assessing relative biomass yields between strains and
treatments can be challenging due to the lack of standardized
practices for measuring biomass yields or net productivity. Algal
biomass yields may be quantified based on optical density, cell
numbers per unit volume, dry weight per unit volume and dry
weight yield per unit land area. Furthermore, since algae are
grown under a variety of environmental conditions it can be
challenging to assess relative yields between different studies.
Given that solar energy inputs are based on the photon flux
density per unit area it is useful to assess biomass yields based on
areal productivity per unit time (gdw/m
2
/day) so that energy
conversion efficiencies can be directly assessed.
Another important consideration in choosing algal strains
for biomass production is the biochemical composition (lipid,
carbohydrate and protein makeup) of the algae. Many algae
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species can also facultatively adjust their relative lipid and
carbohydrate levels depending on culture conditions (Boussiba
et al., 1987;Negi et al., 2016;Sarwer et al., 2022). In a metanalysis
of the biomass composition of the Chlorophyta and Ochrophyta
it was determined that the Chlorophytes had on average 33%
protein, 16% lipid, 14% carbohydrate and 12% ash content per
unit dry weight. In contrast, the Ochrophytes had 33% protein,
21% lipid, 14% carbohydrate, and 19% ash content by dry weight.
Therefore, the ratio of carbohydrate to lipid (0.9) was greater for
the Chlorophytes than for the Ochrophytes (0.5) (Finkel et al.,
2016). Thus, the Ochrophytes were on average more energy
dense than the Chlorophytes.
Recently, one genus of algae, Picochlorum, has emerged as
one of the highest and most stable biomass producers (Dahlin
et al., 2019;Gonzalez-Esquer et al., 2019;Mucko et al., 2020;
Cano et al., 2021;Krishnan et al., 2021). Picochlorum are marine
algae having a wide global distribution that tolerate high salinity
and temperatures (40 C) well allowing for cultivation in a broad
range of environments (Zhu et al., 2008;Dahlin et al., 2019;
Krishnan et al., 2021). Furthermore, recent studies have indicated
that some Picochlorum species have greater photosynthetic
efficiency than many algal species (Cano et al., 2021). This
enhanced efficiency is associated with ability to self-adjust
their light harvesting antenna size to optimize light use
efficiency in dynamically changing light environments
(Perrine et al., 2012;Negi et al., 2020;Cano et al., 2021). In
addition, P. celeri has been shown to be able to both adjust its
light harvesting antenna size as well as accumulate high levels of
photoprotective carotenoids involved in non-photochemical
quenching of excess radiation (Cano et al., 2021). Recently,
molecular toolboxes for the genetic manipulation of P. celeri
have been developed including genetic transformation
(transformation systems and the development of selectable
marker genes) and genome editing tools (Cas9) allowing for
additional engineering opportunities to improve biomass yields.
Thermodynamic modeling of the energy efficiency of
converting solar photons into chemical energy of biomass in
algae indicates that the type of major energy product stored has
an impact on the overall solar to chemical energy conversion
efficiency. Storage of reduced carbon as starch has been shown to
be more energy efficient than lipid storage (Subramanian et al.,
2013;Finkel et al., 2016;Sun et al., 2018;Aketo et al., 2020;
Changko et al., 2020;Negi et al., 2020). The greater energy
storage efficiency of starch versus lipids is largely due to the
loss of previously reduced carbon during lipid synthesis that
occurs with the decarboxylation of pyruvate to produce acetyl
CoA for lipid synthesis. This loss of previously fixed carbon does
not occur during starch accumulation. It follows to reason that
screening techniques for the selection of high biomass
production strains efforts should focus on high starch content
strains rather than high lipid strains.
Recently, a green algal species Pseudoneochloris sp. Strain
NKY372003 was shown to accumulate very high starch levels
relative to other algal strains (68% of total biomass) (Aketo et al.,
2020). In addition, Pseudoneochloris has the ability to increase its
cell volume by over 100-fold. In comparative growth studies, the
volumetric biomass yield (8.1 gdw/L) of Pseudoneochloris was
shown to be 47% greater than for Tetraselmis (5.5 gdw/L) one of
the higher known areal biomass producers (37 gdw/m
2
/day)
(Table 1). These results suggest that alterations in the
allocation of fixed carbon between starch and lipid storage
could substantially impact algal biomass yields. Given the fact
that starch has a substantially higher mass density (1.54 g/cm
3
)
than lipids (0.91 g/cm
3
), simple sedimentation or isopycnic
centrifugation selection systems could be used to efficiently
bioprospect for high starch/high biomass strains. Due to the
greater interest in algal lipids for biofuel production, however,
less research effort has focused on bioprospecting for high starch
containing strains than for high lipid strains.
As previously mentioned, development of management
systems for the control of pathogens in open pond production
systems is only in its infancy (Richmond et al., 1990;Davis and
Laurens, 2010;Olivares et al., 2016;Lammers et al., 2017).
Pathogen/herbivore infections can cause the very rapid in (as
little as 2 days) and complete collapse of algae pond production
systems. A variety of strategies have been used to control
pathogens in algal cultivation systems. Chemical pathogen
management practices for pathogen and herbivores have
TABLE 1 Relative biomass yields from high biomass producing strains of algae.
Species Biomass yield (gdw/m
2
/day) Outdoor growth location
Chlorella sorokiniana (Huesemann et al., 2018) 36 Mesa, AZ, United States
Picochlorum celery (Krishnan et al., 2021)31–40 Mesa, AZ, United States
Picochlorum renovo (Dahlin et al., 2019) 34 Mesa, AZ, United States
Tetraselmis sp.(Laws et al., 1986) 37 Kauai, HI, United States
Scenedesmus UTEX393 (Davis and Laurens, 2010) 27 Mesa, AZ, United States
Nanochloropsis (Boussiba et al., 1987) 25 Israel
Cyclotella sp.(Weissman and Goebel, 1987) 30 California, United States
Chaetoceros muelleri (Weissman and Goebel, 1987) 32 California, United States
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included the use of ammonia as a nitrogen source, phosphite as a
source of phosphorous in strains engineered to oxidize phosphite,
salts, and antibiotics (Lammers et al., 2017;Changko et al., 2020).
Physical management practices include taking advantage of the
differential sheer sensitivity of algae and versus pathogens to
mechanical forces such as pumping or sonication (Lammers
et al., 2017;Marrone et al., 2018). Competing weedy algae and
pathogens can also be controlled using pulsed rather than
continuous applications of macro-nutrients. The greatest
success in managing pond crashes, however, has been achieved
through the selection of algal strains that are naturally resistant to
pathogens (Krishnan et al., 2021;Pang et al., 2022). In large part
the molecular basis for disease resistance is not known.
Interestingly, some species of Picochlorum have been shown to
grow heterotrophically on bacteria presumably by phagocytosis
opening the possibility for breeding or engineering this trait into
algal production strains to control bacterial pathogens (Pang et al.,
2022). Overall, pathogen control in algae remains one of the major
opportunities for increasing in algal biomass yield.
Breeding algae for increased biomass
production
Improvements in terrestrial crop production yields over the
last 80 years can largely be attributed to improved agronomic
practices and the application of advanced genetic breeding
approaches for crop improvement. More recently, molecular
assisted breeding and genomic selection tools have been
applied to crop breeding and have accelerated the
development of advanced crops with desirable properties.
Molecular assisted breeding has been made possible through
the development of low-cost and efficient genome sequencing
tools. In contrast to crop plants, however, there has been little
application of molecular-assisted breeding tools for the genetic
improvement of algae except for the model species
Chlamydomonas. Chlamydomonas is particularly amenable to
breeding for enhanced yields since its dominant (mating type)
generation is haploid, the dominant growth generation for
Chlamydomonas is haploid. Ecotypes of different haploid
mating types have been identified and used for breeding
purposes. Recently, novel recombinant Chlamydomonas
strains having three-fold increases in biomass yield relative to
their parental strains have been generated through genetic
crosses and strong selection regimes (Lucker et al., 2022). The
increases in yield in the progeny were correlated with widespread
alterations in gene frequencies between the two parental strains.
Unlike Chlamydomonas, however, the dominant generation
for many algae is diploid. Thus, the challenge for mating many
algae strains is how to induce gametogenesis and conjugation.
Induction of meiosis, gamete formation and subsequent mating
in diploid algae is currently an inexact science. The most
common approach to induce meiosis in algae is nutrient
stress in the dark (Huang and Beck, 2003;Přibyl P Light is a,
2013). In addition, exposure of gametes to a short pulse of blue
light activates the phototropin receptor and can induce mating in
some algal strains (Pfeifer et al., 2010). One approach to assess
the effectiveness of various environmental treatments on
FIGURE 1
Changes in apparent DNA ploidy per cell following induction of meiosis, gamete formation and mating in Chlorella sorokiniana Cs-1228. Panel
on the left shows changes in fluorescence levels (FL2-A) of a DNA binding dye per cell following different periods (0, 2, 6 and 22 h) of exposure in the
dark to growth media lacking nitrogen followed by blue light treatment where indicated. The panel on the right shows the presence of flagella in cells
grown for 6 h without nitrogen in the dark and the conjugation of cells and cytoplasmi c exchange 6 h after minus nitrogen treatment in the dark
plus blue light (Dept, 2017).
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gametogenesis and mating is tracking changes in DNA ploidy
levels per cell in real time. This can be accomplished by
quantifying relative changes in cellular DNA content using
DNA binding fluorescent dyes coupled with fluorescent
activated cell counting (Dept, 2017). As shown in Figure 1,
Chlorella sorokiniana Cs-1228 cells starved of nitrogen in the
dark for 6 h had half the DNA content of cells grown in nitrogen
replete media indicative of the production of gametes under
nitrogen stress (Dept, 2017). Gamete induction was observed in
as little as 2 h after transfer of cells to media without nitrogen in
the dark (Figure 1). At 22 h after nitrogen deprivation in the dark
there was a small shift to higher DNA content/cell suggesting
some level of mating and production of diploid cells. But the
additional treatment of cells with a brief pulse of blue light
(1–2 µmol photons/m
2
/s for 5 min) resulted in a near complete
shift to diploid cells indicative of mating. The conversion of
diploid to haploid cells competent for mating during dark
nutrient stress treatments was further supported by the
appearance of flagella required for mating at cells 6 h after
treatment in the dark without nitrogen (Figure 1). Mating was
also demonstrated by cytoplasmic exchange in blue-light treated
cells (Figure 1)(Dept, 2017).
The development of procedures to induce gametogenesis
and mating in other polyploid algae opens the door for the use
of advanced genome wide association studies, molecular
breeding tools and selection for enhanced performance
characteristics and biomass yield (Přibyl P Light is a, 2013;
Lucker et al., 2022). Overall, it is expected that the potential to
increase biomass yields is great using breeding approaches
given the vast genetic diversity that exists in natural algal
ecotypes and the possibility to engineer algae (Baker et al.,
2007;Ng et al., 2017;Crozet et al., 2018;Naduthodi et al.,
2020;Ng et al., 2020;Shokravi et al., 2021).
Directed and adaptive evolution of algae
for increased biomass yield
Biomass production can also be optimized for particular
environments using directed or adaptive evolution approaches.
When coupled with the generation of mutant populations for
selection, additional improvements in yield resulting from new
genotypes may also be achieved. Significantly, additional
understanding of the metabolic factors that regulate growth
can be achieved through comparative genomic,
transcriptomic, proteomic and metabolomic studies of
improved strains selected through directed evolution. For
example, Haematococcus pluvialis strains selected for
improved growth under high (15%) CO
2
conditions were
shown to have enhanced photosynthesis and glycolysis
associated with the increased expression of genes involved in
photosynthetic electron transport including PetH
(ferredoxin–NADPC reductase) and ATPF0A (F-type H
+
-
transporting ATPase subunit), and genes involved in
respiration including, the PetJ (cytochrome c6) gene involved
in NADPH generation. Additionally, genes of the C3 and
TABLE 2 Transgenic modifications of green algae for enhanced biomass production.
Host Target gene Phenotype Cultivation system Biomass
increase
relative
to wild type
Chlamydomonas (Negi
et al., 2020)
Light-regulated chlorophyll a
oxygenase expression mediated by
NAB1 translational repressor
Small light harvesting antenna when
grown in high light and large light
harvesting antenna when grown in low
light
Simulated diurnal pond environment
in PBR
100–180%
Chlamydomonas
(Perrine et al., 2012)
Reduced chlorophyll a oxygenase
expression
30% reduction in antenna size, Chl a/b
ratio = 5 for transgenic vs. 2.2 for wild
type
Simulated diurnal pond environment
in PBR
40%
Chlamydomonas
(Shokravi et al., 2021)
Overexpression of activated
NAB1 gene repressor of LHCII
expression
17% reduction in antenna size. Chl a/
b= 2.2 for transgenic vs. 2.0 for wild
type
PBR supplemented with 3% CO
2
and a
continuous light intensity of 700 µmol
photons/m
2
/sec
30%
Chlamydomonas
(Beckmann et al.,
2009)
Reduced global expression of LHC
family members
30% reduction in Chl content/cell, Chl
a/b ratio = 4
Photoheterotrophic growth in presence
of acetate under continuous illumination
at 1,000 µmol photons/m
2
/sec
Not determined,
but 45% faster
growth rate
Chlamydomonas
(Mussgnug et al., 2007)
Phototropin knock out mutants Global alterations in photosynthesis,
cell cycle control and carbon allocation
Simulated diurnal pond environment
in PBR
100%
Phaeodactylum (Fu
et al., 2017)
Expression of eGFP in the cytoplasm Increased light use and reduced NPQ Simulated outdoor growth in PBR
supplemented with 1% CO
2
50%
Chlorella (Yang et al.,
2017)
Overexpression of cyanobacterial
CBB Cycle enzyme fructose 1,6-
bisphosphate aldolase
1.3-fold higher aldolase activity Low light (40 µmol photons/m
2
/sec)
intensity shaker flasks
6%
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C4 photosynthetic pathways in plants were overexpressed in
improved strains including PddK, pyruvate, orthophosphate
dikinase and FBA, fructose-bisphosphate aldolase. These genes
facilitated carbon uptake under high CO
2
growth conditions (Li
et al., 2017). Among the greatest improvements in biomass yield
was achieved using directed evolution approaches under high
CO
2
growth conditions.. As much as a 3-fold increase in algal
biomass production (3.7 gdw/L) was achieved after 31 rounds of
selection under high (10–20%) CO
2
(Li et al., 2015). This increase
in biomass yield was associated with the accumulation of greater
amounts of starch per cell. It is important to note however, that
the molecular adjustments resulting in increased biomass yields
may not be the same for slow growing versus fast growing
parental strains. If the biomass yield of the parental strain is
low to begin with, modest metabolic changes resulting in large
relative increases in biomass yield may not be applicable to very
fast-growing strains. The metabolic bottlenecks limiting growth
for each strain must be evaluated.
Engineering algae for increased biomass
yield
Metabolic engineering strategies to increase biomass yields
can be separated into at least four different categories including
increasing carbon source strength, increasing carbon sink
strength, alterations in the expression of master regulatory
genes that control global carbon partitioning and flux rates,
and strategies that control the cell cycle and cell division rates.
First, we describe genetic manipulations that increase carbon
source strength or rates of photosynthesis (Table 2).
Based on relative thermodynamic efficiency, the earliest steps
in photosynthesis involved in light capture and utilization,
i.e., the conversion of chlorophyll excited states into charge
separated states driving photosynthetic electron transfer
processes, are collectively the least efficient processes in
biomass production (Subramanian et al., 2013;Okada et al.,
2020;Vecchi et al., 2020). This is largely attributed to the fact that
photosynthetic electron transfer rates saturate at 20–25% of full
sunlight intensity in all algae and plants (Perrine et al., 2012).
This is due to the fact that at full sunlight intensities the rate of
photon capture by the light harvesting complexes is 5- to 10-fold
faster than the rate-limiting steps in photosynthetic electron
transport, i.e., the oxidation of plastoquinol by the
cytochrome b6f complex (Baker et al., 2007;Perrine et al.,
2012). In addition, as the proton gradient increases across the
thylakoid membrane following illumination rates of plastoquinol
oxidation are further diminished (Baker et al., 2007;Kiirats et al.,
2009).
When addressing electron transport or metabolic steps
having rate-limiting kinetics one strategy to alleviate
bottlenecks is to overexpress the enzyme(s) or enzyme
complex that are rate limiting. For example, to compensate
for the slow rate of RuBP carboxylase/oxygenase (RuBisCO)
catalysis plants and algae substantially overexpress the enzyme
RuBisCO). In algae the cytochrome b6f complex, catalyzes the
rate-limiting step in electron transport. The cytochrome b6f
complex is present in equal stoichiometry with the
photosystem I and II reaction center complexes (Kiirats et al.,
2009). Thus, it is conceivable that elevating the relative number of
cytochrome b6f complexes coupled with accelerated dissipation
of the proton gradient by the ATPase complex, could compensate
for the slow rate of plastoquinol oxidation. To date, however,
there have been no reports of successfully overexpressing the
cytochrome b6f complex to address this bottleneck in electron
transport. This inability to substantially alter the stoichiometry of
cytochrome b6f complexes may in part be due to the unique
requirements for assembling stable cytochrome b6f complexes.
An alternative strategy for addressing inefficiencies in
coupling chlorophyll excited state transitions to electron
transport rates has been to reduce the optical cross-section or
size of the light harvesting antenna to a level where the rate of
photon capture corresponds with the rate of electron transfer.
Reductions in light harvesting antenna size have been achieved
by multiple means in mutant and engineered Chlamydomonas.
One of the more successful strategies for reducing light
harvesting antenna sizes has been reduction in chlorophyll b
(Chl b) accumulation. Chl bis present only in the peripheral,
nuclear-encoded, light harvesting pigment/protein complexes
associated with reaction center complexes (photosystem II and
I) and is not present in the chloroplast-encoded reaction center
proximal antenna proteins or the core reaction center complex
proteins. The core reaction center proteins only contain only Chl
a(Melis, 2009;Perrine et al., 2012). Reduction in Chl blevels
destabilizes the light harvesting protein subunits resulting in the
turnover of Chl b-binding proteins effectively reducing the
peripheral light harvesting antenna size (Melis, 2009;Perrine
et al., 2012;Negi et al., 2020). The earliest studies on reduction of
Chl blevels in algae were those carried out by Melis and
colleagues (Melis, 2009), Their studies on antennae size
modulation focused on the selection for mutants that did not
accumulate Chl b. When grown under photoheterotrophic
conditions the Chl b-less mutants showed some improvements
in yield. However, when grown strictly photosynthetically
chlorophyll aoxidase mutants, unable to produce any Chl b,
had substantially reduced growth rates compared to their wild-
type parents (Perrine et al., 2012).
But all things in moderation. By fine tuning Chl blevels by
partially but not completely suppressing chlorophyl aoxygenase
(CAO) activity it was determined that moderate reductions in
light harvesting antenna size resulted in the greatest increases in
photosynthetic efficiency. The highest biomass producing strains
were shown to have lost one LHCII trimer complex relative to
wild-type algae, equivalent to a Chl a/b ratio of 5 (Perrine et al.,
2012). Transgenic algae having Chl a/b ratios lower than 5 (larger
antenna sizes) including wild-type algae (Chl a/b = 2.0–2.5) or
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algae having Chl a/b ratios greater than 5 (smaller antenna) had
reduced photosynthetic rates and reduced abilities to tolerate
high light stress due to a reduced capacity for non-photochemical
quenching of excess captured energy. Significantly, biomass
yields for algae having Chl a/b ratios of 5 was 40% greater
than those of wild-type algae when grown in photobioreactors
simulating outdoor pond cultivation conditions (Perrine et al.,
2012). Similar observations were made for plants engineered to
have reduced light harvesting antenna sizes. Optimal biomass
production in Camelina was achieved in plants having a Chl a/b
ratio of 5 (Friedland et al., 2019).
Reductions in light harvesting antenna complexes have also
been achieved by overexpression of the NAB1 protein which
regulates the expression of multiple light harvesting complex
protein (Beckmann et al., 2009). The NAB1 protein is a
translational repressor that binds to the 5′end of transcripts
encoding members of the light harvesting complex.
Overexpression of the activated (reduced disulfides)
NAB1 protein resulted in a 17% reduction in antenna size in
transgenic Chlamydomonas corresponding to a Chl a/b ratio of
2.2. Biomass yields were 30% greater in NAB1 transgenics
relative to the parental wild-type strain when grown in
photobioreactors supplemented with elevated CO
2.
Additional
approaches have been used to engineer light harvesting antenna
size. Mussgnug et al (Mussgnug et al., 2007) described a more
generalized approach to reduce light harvesting antenna size by
expressing an RNAi construct that targeted a common DNA
sequence present in the 5′untranslated region of multiple LHCII
family members. They observed a 30% reduction in Chl content/
cell leading to a Chl a/b ratio of 4. Under mixotrophic growth
conditions the transgenics had a 45% faster growth rate than wild
type, however, no biomass studies were reported.
To date, the greatest biomass increases resulting from genetic
engineering approaches have been achieved through the light
regulation of light harvesting antenna size (Negi et al., 2020).
Dynamic regulation of light harvesting antenna size was achieved
by co-expression of a light-regulated NAB1 translational
repressor along with coexpression of a modified CAO gene
having a 5’NAB1 binding light response element expressed in
aCAO knock strain. In contrast, under high light conditions light
harvesting antenna sizes were reduced due to increased
NAB1 expression. In contrast, under low-light growth
conditions antenna sizes were increased due reduced
NAB1 expression in transgenic strains. It was also observed
that sensitivity to photoinhibition was reduced relative to wild
type cells in the transgenic strains relative to wild type.
Another novel approach to alter light harvesting efficiency has
been the expression of light absorbing proteins in the stroma of
chloroplasts. The expression of the green fluorescent protein in the
chloroplasts of the diatom Phaeodactylum tricornutum resulted in
a 50% increase in biomass yield (0.23 gdw/L/day) under simulated
pond growth conditions supplemented with 1% CO
2
(Fu et al.,
2017). This increase in biomass yield was only observed when the
transgenics were grown under light intensities (400 µmoles
photons/m2/sec) that were greater than those that light-saturate
photosynthetic electron transport. Interestingly, non-
photochemical quenching in GFP expressing strains was
reduced under high-light growth conditions relative to wild
type. These results suggest that GFP was shading the light
harvesting apparatus resulting in reduced energy dissipation
through NPQ allowing for enhanced photosynthetic electron
transport at high light levels.
One of the more elegant strategies to enhance algal biomass
production has been the genetic manipulation of master growth
regulatory genes that globally regulate multiple genes involved in
biomass accumulation. One such algal regulatory gene is the blue
light photoreceptor, phototropin (Im et al., 2006;Pfeifer et al.,
2010;Petroutsos et al., 2016). In Chlamydomonas phototropin
regulates the expression of multiple gene systems including genes
involved in the sexual life cycle, eye spot size, and genes involved
chlorophyll, carotenoid, and chlorophyll binding protein
synthesis (Im et al., 2006;Petroutsos et al., 2016). Negi et. al.
(Negi et al., 2017) demonstrated that phototropin knock out
mutants had higher Chl a/b ratios than wild type cells ranging
from 2.9 when grown under low light to 3.4 when grown under
high light conditions associated with a reduction in light
harvesting antenna size. In addition, cell division rates and
biomass accumulation rates were substantially increased in
phototropin knock out mutants. RNAseq experiments
indicated that the expression of multiple genes involved
photosynthesis, carbon metabolism, and cell division rates
were significantly altered in phototropin mutants. Phototropin
mutants had a 2- to 5-fold increase in the expression levels of the
Rieske Fe-S protein (cytochrome b6f complex), the Calvin-
Benson-Bassham Cycle (CBBC) enzymes ribulose-1,5-
bisphosphate carboxylase/oxygenase, sedoheptulose
1,7 bisphosphatase glyceraldehyde-3- phosphate
dehydrogenase, carbonic anhydrase, and genes involved in
starch synthesis including ADP glucose pyrophosphorylase,
starch synthase, and genes involved in respiration and fatty
acid biosynthesis In addition, the SNRK1 gene that shares
homology with the major growth regulatory genes KIN10 and
KIN11 in plants had ten-fold higher expression levels in
phototopin mutants (Baena-González et al., 2007;Negi et al.,
2017). Genes involved in cell cycle control including NIMA
(never in mitosis), NEK2, NEK6 (NIMA related kinases),
RCC1 (regulator of chromosome condensation, cyclin and
cyclin-dependent kinases: Cyclin-dependent kinases, and
MAT3 a homolog of retinoblastoma protein (MAT3/RB) were
also upregulated 2-to 15-fold in photoptropin mutants relative to
wild type. Overall, there was a two-fold increase in biomass yield
in phototropin mutants relative to wild-type cells when grown
under environmental conditions that mimicked outdoor light
and temperature conditions (Negi et al., 2017). However, there is
some evidence that phototropin mutants may be more
susceptible to photoinhibition than wild type (Li et al., 2017)
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These results indicate that the coordinated regulation of source
and sink strength genes as well as cell cycle control genes can
enhance biomass accumulation in algae.
In contrast to efforts to improve biomass yields through
alterations in light use efficiency, there have been limited efforts
to improve algal biomass yields through manipulation of CBBC
enzymes or by increasing carbon sink strength. There have,
however, been substantial efforts to increase lipid
accumulation in microalgae through genetic engineering and
genome editing approaches often at a loss in biomass
accumulation (Gonzalez-Esquer et al., 2019;Fayyaz et al.,
2020;Ng et al., 2020;Shokravi et al., 2021). One approach to
increase biomass accumulation through manipulation of the
CBBC activity was achieved through enhancement of fructose
1,6-bisphosphate aldolase activity, an enzyme playing a major
role in carbon flux control in the CBBC (Yang et al., 2017).
Transgenic Chlorella cells overexpressing fructose 1,6-
bisphosphate aldolase were shown to have 20% higher rates of
photosynthesis but only a 6% increase in biomass yields relative
to wild type. The small increase biomass yields may be due to the
low light conditions under which the algae were cultivated.
Additionally, it has been demonstrated that genetic
complementation of Chlamydomonas mutants impaired in
starch synthesis (ADP glucopyrophosphorylase mutants)
resulted in reduced generation of potentially toxic reactive
oxygen species indicating the critical role starch synthesis
plays as an electron sink (Saroussi et al., 2019). Finally, starch
hyperaccumulation mutants impaired in glycolysis were shown
to have 72% higher cell numbers at stationary phase than wild-
type cells (Koo et al., 2017). Collectively, these results
demonstrate the critical role carbon metabolism and starch
accumulation plays in overall algal biomass accumulation.
Finally, biomass yields are also determined by cell cycle
kinetics and changes in cell volume (Li et al., 2016). The
regulation of cell cycle activity and cell size in algae, however,
is less well understood than central metabolism but offers
significant opportunities for manipulation of biomass
production (Li et al., 2016).
Conclusion
Microalgae are gaining increased attention as possible
alternative food sources, platforms to produce high value
co-products, biofuels, and for long-term carbon
sequestration in geologic formations. Recent life cycle
analyses of algal production systems have determined that
algal biomass production rates are one of the major
bottlenecks impeding the commercialization of algal
biomass, bioproducts and biofuel products. Thus, the need
for enhanced algal biomass production to make algae
commercially competitive with other biomass production
platforms.
Theoretical and empirical evidence indicate that algae that
accumulate starch as their major reduced energy reserve have
faster growth rates and greater biomass accumulation potential
than algae that predominantly store lipids as energy reserves.
Since carbon skeletons for lipid production are in large part
derived from starch it is apparent that the focus on identifying
algal strains that are high lipid accumulators may not be the most
effective bioprospecting strategy for identifying algal strains that
have higher biomass yield. A potentially better strategy for
identifying new commercials strains of algal for coproduct
production may be to identify the highest starch accumulating
species.
Substantial progress has, however, been made in identifying
algal strains that have stable, long-term biomass production in
outdoor environments. Genome sequencing and the development
of molecular toolboxes including genetic transformation,
enhanced and inducible gene expression cassettes, and genome
editing tools will substantially expand the potential of algae as
sustainable platforms for the production of high value
biomolecules and for carbon sequestration. Progress has been
made in improving light use efficiency in photosynthesis
resulting in nearly a 200% increase in biomass yield in
simulated outdoor growth studies. Surprisingly, there has been
less research focus on increasing carbon flux through the CBBC or
increasing carbon sink strength to improve biomass yields.
Perhaps, the greatest unrealized potential for improvements
in algal biomass yields is through the application of molecular
assisted breeding of algae. With the increased availability of
annotated algal genomes, isolation of ecotypes from diverse
environments, a growing understanding of methods to induce
gametogenesis and mating and the availability of high
throughput screening and selection systems for identifying fast
growing progeny of sexual crosses the future for algae having
enhanced biomass yields is bright.
Author contributions
RS drafted the review article. SS contributed the sections on
Chlorella sorokiniana mating and Figure 1.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
Frontiers in Energy Research frontiersin.org09
Subramanian and Sayre 10.3389/fenrg.2022.979747

organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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