Environmental Science and Pollution Research (2023) 30:79654–79675

https://doi.org/10.1007/s11356-023-28123-6

REVIEW ARTICLE

A comprehensive review on microbial lipid production from wastes:

research updates and tendencies
Haishu Sun1,2 · Zhen Gao1 · Lirong Zhang3 · Xiaona Wang1,2 · Ming Gao1 · Qunhui Wang1,3

Received: 29 December 2022 / Accepted: 1 June 2023 / Published online: 16 June 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Microbial lipids have recently attracted attention as an intriguing alternative for the biodiesel and oleochemical industries
to achieve sustainable energy generation. However, large-scale lipid production remains limited due to the high processing
costs. As multiple variables affect lipid synthesis, an up-to-date overview that will benefit researchers studying microbial
lipids is necessary. In this review, the most studied keywords from bibliometric studies are first reviewed. Based on the results,
the hot topics in the field were identified to be associated with microbiology studies that aim to enhance lipid synthesis and
reduce production costs, focusing on the biological and metabolic engineering involved. The research updates and tendencies
of microbial lipids were then analyzed in depth. In particular, feedstock and associated microbes, as well as feedstock and
corresponding products, were analyzed in detail. Strategies for lipid biomass enhancement were also discussed, including
feedstock adoption, value-added product synthesis, selection of oleaginous microbes, cultivation mode optimization, and
metabolic engineering strategies. Finally, the environmental implications of microbial lipid production and possible research
directions were presented.

Keywords Single cell oil · Feedstock · Oleaginous microbe · Cultivation mode · Metabolic engineering · Environmental
implication

Introduction as promising substitutes for the oleochemical industry in a

Global climate change and other environmental problems
resulting from the excessive consumption of fossil fuels
are urgent concerns (Kim et al. 2019; Sundaramahalingam
et al. 2022). Faced with a shortage of petrochemical petro-
leum, the contradiction of food for oil, and deteriorating
environmental problems, it is urgent to develop renewable,
clean, and environmentally friendly energy sources such as
biofuels (Park et al. 2019). Microbial lipids have emerged
Responsible Editor: Ta Yeong Wu
* Xiaona Wang
wangxiaona071@163.com
1
School of Energy and Environmental Engineering,
University of Science and Technology Beijing,
Beijing 100083, China
2
Shunde Innovation School, University of Science
and Technology Beijing, Foshan 528399, China
3
Tianjin College, University of Science and Technology,
Beijing, Tianjin 301811, China
move toward renewable and sustainable energy production
(Chatterjee and Mohan 2020; Karamerou and Webb 2019).
Microbially derived lipids are particularly attractive because
of their high quality, shorter cultivation time, independ-
ence from geographical or climatological conditions, lower
space requirements, and non-competitiveness with food sup-
ply (Chatterjee and Mohan 2020; Tomas-Pejo et al. 2021).
However, large-scale microbial lipid development is limited
by high processing costs and high prices of substrates (Cho
and Park 2018). Therefore, the exploration of cost-effective
feedstocks, combined with the use of waste substrates, which
are environmentally and economically sustainable, might be
a feasible alternative for reducing costs (Tan et al. 2022).
Oleaginous microbes can utilize low-cost feedstocks
and waste substrates, such as lignocellulosic biomass and
agro-residues, for higher lipid production (Garlapati et al.
2020). Oleaginous properties are inherent to certain spe-
cies of yeasts, microalgae, molds, and bacteria (Khot et al.
2020), and the identification of excellent oleaginous strains
is a prerequisite for the industrialization of microbial lipids.
Industrial production strains should be robust, stable, and

13
Vol:.(1234567890)
Environmental Science and Pollution Research (2023) 30:79654–79675
highly productive, with the ability to assimilate a diverse
array of carbon (C) sources and be tolerant to a wide range
of inhibitors in complex substrates (Chaturvedi et al. 2019).
In addition to the adoption of inexpensive raw materials,
the bioconversion of feedstock into value-added products
could add value to fermentation processes. Therefore, a co-
production system of microbial lipids with valuable com-
pounds, such as pigments and polyunsaturated fatty acids
(PUFAs), not only improves the utilization of resources but
also enhances the economics of microbial lipids (Chen et al.
2021; Manowattana et al. 2018).
To increase the economic competitiveness of the micro-
bial lipid manufacturing process, cultivation modes have
been optimized, and metabolic pathways have been modified
(Chen et al. 2021). Although microorganisms have inherent
metabolic pathways for the synthesis of microbial lipids, the
theoretical yield of microbial lipids is limited to approxi-
mately 0.2–0.3 g/g, which hinders industrial production
and commercialization (Chen et al. 2021; Das et al. 2020).
Biomass growth is the predominant process when C and N
are abundant; however, microbial lipids are generally syn-
thesized under the prerequisites of sufficient C and limited
nutrients (N, P, or S) (Ma et al. 2018). Therefore, optimizing
cultivation modes is crucial because of the distinct nutri-
ent demands of the two stages. Maximum lipid yield occurs
when minimal C flux is dedicated to the biomass (Yan and
Pfleger 2020). Metabolic engineering approaches can manip-
ulate the microbial C-energy balance and enhance the yield
and titer of lipids (Mehmood et al. 2021). Metabolic engi-
neering involves modifying the genetics and metabolism of
microorganisms toward the desired biomass composition,
which can increase the titer, rate, and yield of the targeted
metabolite (Kim et al. 2019). The overexpression of target
enzymes and blockage of competitor pathways are two key
metabolic engineering methods for boosting yield (Das et al.
2020; Yan and Pfleger 2020).
Fig. 1  Number of publications
on microbial lipids during the
past 21 years (2000–2021)
79655
Owing to the crucial role of microbial lipids in promoting
environmental and economic sustainability, it is not surpris-
ing that more than 3000 articles have been published on
this topic over the last 2 decades (Fig. 1). However, most
review papers on microbial lipids focus on the effects of
one or two factors on lipid production, and hardly any of
these reviews provide comprehensive recommendations for
enhancing lipid biosynthesis (Park et al. 2019; Saha and
Mukhopadhyay 2021). As multiple factors can affect lipid
production, an up-to-date review that will benefit researchers
studying microbial lipids is required. This review aimed to
summarize the research hotspots in microbial lipids, includ-
ing feedstocks, oleaginous microbes, cultivation modes,
research methods, and products. In particular, feedstock and
associated microbes, as well as feedstock and correspond-
ing products, were thoroughly evaluated. Recent advances
and tendencies of the most studied keywords in research on
microbial lipids were also discussed in depth, and an over-
view of the cultivation modes and metabolic engineering
approaches to maximize microbial lipid production is also
presented. Finally, the environmental implications of micro-
bial lipid production and possible research directions were
discussed. Overall, this study aimed to provide an overview
of the current status of research in the field of microbial lipid
production and inspire further innovation in this area.
Research tendencies analysis based
on bibliometrics
In this study, keywords (“microb* lipid*” or “microb* oil*”
or “single cell oil” or “single cell lipid*” or “oleaginous
micro*” or “oleaginous algae*” or “oleaginous yeast” or
“oleaginous bacteria*” or “oleaginous mold” or “oleaginous
fung*”) were used as topic search phrases to acquire all the
index of articles published in the Web of Science database
13
79656
from 2000 to 2021. The research on microbial lipids started
relatively late, with only a few publications per year until
2008. However, since 2010, the number of articles on this
topic has substantially increased, likely due to several factors
such as the emergence of active clinical and safety trials as
well as the establishment and consolidation of organizations
engaged in the microbial lipids production (Palazzolo and
Garcia-Perez 2022). A noteworthy trend is the fact that over
300 papers were published in 2021 alone, indicating a sig-
nificant increase in researchers’ interest in microbial lipids.
The keywords associated with research on microbial
lipids include feedstocks, oleaginous microbes, products,
research methods, cultivation modes, and products, as
illustrated in Fig. 2. Among these, oleaginous microbes
and products have received the most attention (Fig. 2a). In
Fig. 2  Keywords analysis of research on microbial lipids
13
Environmental Science and Pollution Research (2023) 30:79654–79675
terms of feedstock (Fig. 2b), the most frequently used carbon
source is glucose. Various waste substrates have been inves-
tigated due to their low cost, with lignocellulose being the
most commonly researched feedstock. To enable the microbe
to access the nutrients found in waste substrates, appropri-
ate pretreatment methods such as enzymatic hydrolysis
are required (Cai et al. 2016). Additionally, glucose-based
wastes such as molasses have been utilized for lipid produc-
tion. Among the oleaginous microbes (Fig. 2c), yeast had
the highest occurrences, followed by microalgae, molds, and
bacteria. Proper cultivation modes are essential to promote
high lipid generation by supplying the essential nutrients
at the appropriate time (Karamerou and Webb 2019). The
batch mode is a well-studied and fundamental approach for
lipid production (Fig. 2d). Given that microbes compete for
Environmental Science and Pollution Research (2023) 30:79654–79675
C sources in the two primary processes of cell proliferation
and lipid accumulation, metabolic pathways of microbial
lipids have gained increasing attention. Many studies have
tried to regulate C flow to produce lipids through research
methods such as metabolic engineering tools (Fig. 2e).
For product generation (Fig. 2f), one significant func-
tion of microbial lipids is their potential as substitutes for
valuable lipids, such as cocoa butter. This function is almost
impossible to achieve through “de novo” lipid production
because in many cases, the lipid composition of the micro-
organism remains relatively stable (Huang et al. 2013). For
example, the reserve lipids of Y. lipolytica can serve as a
substitute for cocoa butter due to the use of an oleic acid
donor as a co-substrate with stearin (Papanikolaou and
Aggelis 2010). Synthetic biology and metabolic engineer-
ing have enabled increased triacylglycerol (TAG) production
in oleaginous yeasts, such as Y. lipolytica and S. cerevisiae,
providing a potential solution for the production of cocoa
butter substitutes via “de novo” lipid accumulation path-
ways (Konzock et al. 2022; Wang et al. 2020b). Owing to
their high carbon-to-heteroatom ratios, microbial lipids are
desirable feedstocks for the production of renewable fuels.
These TAGs contain fatty acids that are suitable for bio-
diesel production (Kosa and Ragauskas 2011). Biodiesel,
similar to other renewable energy sources, has a high energy
density and is nontoxic, biodegradable, and environmentally
friendly (Zhang et al. 2021a). Additionally, the production of
high-value products such as docosahexaenoic acid (DHA),
eicosapentaenoic acid (EPA), carotenoids, and arachidonic
acid (ARA), has garnered increasing interest (Jakhwal et al.
2022).
Feedstock
Feedstock and associated microbes
Oleaginous microbes can use hydrophilic and hydrophobic
substrates to accumulate lipids via two pathways: de novo
and ex novo lipid synthesis. It is noteworthy that both of
these pathways offer distinct advantages: de novo lipid fer-
mentation can generate a considerable quantity of lipids,
while ex novo lipid fermentation can modify the lipid com-
positions to satisfy the requirements of the chemical or food
industries (Huang et al. 2017). Together, these two strategies
provide a powerful platform for the production of various
lipids with different properties and applications.
“De novo” lipid fermentation
Oleaginous yeasts are among the most widely studied
microbes for lipid production, due to their ability to con-
sume a broad range of low-cost materials (Fig. 3). Relatively
79657
Fig. 3  Frequently studied feedstocks and their associated microbes
for lipid production
few studies have been conducted on feedstocks and their
associated bacteria for lipid production, as they generally
do not retain substantial amounts of lipids (Palazzolo and
Garcia-Perez 2022). Oleaginous yeasts such as Lipomyces,
Rhodotorula, and Rhodosporidium are particularly note-
worthy for their ability to rapidly consume diverse organic
materials and stably produce lipids, making them a popular
choice for microbial lipid production. Lipomyces can convert
hemicellulosic hydrolysate sugars into lipids without prior
detoxification, making it a promising candidate for lipid
generation in the presence of toxic components (Qin et al.
2017). Furthermore, inhibitors are depleted throughout the
fermentation process, probably due to yeast metabolism, as
the Lipomyces genome contains key genes involved in inhib-
itor metabolism. Rhodotorula is a ubiquitous yeast species
used in the manufacture of natural carotenoids (Gujjala et al.
2019). Rhodosporidium, another yeast genus, plays a vital
role in the environment and is beneficial as many species can
convert lignocellulosic biomass into lipids. Yarrowia prefers
oil- and food-rich conditions. The genome of the model Yar-
rowia lipolytica was sequenced, which could facilitate syn-
thetic biology and genetic engineering applications to boost
its lipid production performance (Madzak 2018).
Glucose is the feedstock most commonly used by ole-
aginous microbes to produce lipids because it is the most
suitable carbon source for microbial growth. However, the
cost of large-scale lipid production using glucose as a raw
material is high; therefore, agro-industrial and municipal
wastes, as well as other non-food raw materials, are being
used to explore the feasibility of microbial lipid production.
Microbial lipid production from wastewater with high
levels of organic matter has been found to be slightly more
13
79658
prevalent using yeasts (Singh et al. 2022). Cryptococcus
such as Cryptococcus curvatus, Cryptococcus Laurentis, and
Cryptococcus podzolicus were frequently used for simulta-
neous wastewater treatment and microbial lipid production
(Arous et al. 2019; Santos et al. 2014). Cryptococcus cur-
vatus had higher lipid production than Y. lipolytica in media
made from primary wastewater (Chi et al. 2011).
A suitable feedstock for biodiesel production is food
waste (Carmona-Cabello et al. 2021), a significant munici-
pal solid waste with a high organic matter content (Xu et al.
2022; Zhang et al. 2021c), rich in carbohydrates, proteins,
and starch (Ma et al. 2022). Rhodosporidium, Cryptococ-
cus, and Trichosporon were the most frequently studied gen-
era. Trichosporon differs from other yeasts for the ability to
use highly diverse C sources, and it has a high tolerance to
5-hydroxymethylfurfural (5-HMF), which is present in most
food waste (Arous et al. 2019). Our group has conducted
extensive studies on the microbial lipid production from
food waste. Rhodosporidium toruloides 2.1389 can use food
waste saccharified liquid as a substrate for lipid production
without the addition of external nutrients (Ma et al. 2019).
A variable pH control strategy was applied to the same ole-
aginous strain, resulting in a lipid yield of 11.10 g/kg of food
waste (Gao et al. 2019).
Lignocellulose, which is present in agricultural and
agro-industrial residues, is the most abundant sustainable
resource for the generation of advanced biofuels (Ruan
et al. 2013). Biomass is mainly composed of three poly-
meric components: cellulose, hemicellulose, and lignin.
As most oleaginous microbes lack cellulase and hemi-
cellulose activities, lignocellulosic biomass is typically
subjected to hydrolysis to convert its polymers into fer-
mentable sugars, which can be used as a carbon source
for lipid production by oleaginous microbes (Santek et al.
2018). However, the effective conversion of lignocellu-
losic sugars into high-quality lipids remains a significant
issue for oleaginous yeasts as they preferentially consume
glucose instead of other sugars present in lignocellulosic
hydrolysates (Qin et al. 2017). Despite these challenges,
several studies have investigated the effectiveness of ole-
aginous yeasts in utilizing lignocellulosic hydrolysates for
lipid production. Recently, significant lipid titers (39–45%
of the theoretical yield) have been observed for several
strains of oleaginous yeasts cultivated on non-detoxified
lignocellulosic hydrolysates. Yarrowia lipolytica cultivated
on hydrolysates of defatted rice bran had a lipid content of
48.02% (Tsigie et al. 2012). Fei et al. (2016) investigated
various fed-batch modes to optimize lipid production by
Rhodosporidium toruloides using corn stover hydrolysate.
Based on their findings, online sugar control was the most
efficient approach, resulting in the highest lipid content
(59%). Rhodotorula babjevae has demonstrated remark-
able potential as a fermentative strain for the conversion of
13
Environmental Science and Pollution Research (2023) 30:79654–79675
wheat straw hydrolysates into lipids. In batch cultivation,
this strain had a high lipid content of 18.1 g/L and a high
yield of 0.24 g/g carbon source (Brandenburg et al. 2021).
Cheesy whey, a major byproduct of cheese manufacture
in the dairy industry, is an appealing substrate for micro-
bial lipid production because of its accessibility and high
organic load (Chan et al. 2018). Various yeast strains, such
as Candida sp., Cryptococcus sp., and Apiotrichum cur-
vatum, have been found to be capable of producing large
amounts of lipids when cultured in shake flasks. Carota
et al. (2017) used ricotta cheese whey as a substrate for
microbial lipid generation in a shake flask and observed
that Cryptococcus curvatus and Cryptococcus laurentii
produced substantial quantities of lipids (6.8 and 5.1 g/L,
respectively). Furthermore, using cheese whey for cocoa
butter substitute production is almost economically viable.
A cocoa-butter substitute was synthesized from cheese
whey using Apiotrichum curvatum ATCC 20509 culti-
vated in a batch bioreactor (Davies et al. 1990). The pri-
mary disaccharide in cheesy whey, lactose, is hydrolyzed
by acid or an enzyme to produce glucose and galactose.
Tchakouteu et al. (2015a) observed that lactose acted as
a substrate that facilitated the accumulation of lipids by
Cryptococcus curvatus. Mucor circinelloides was used to
produce lipids on cheese hydrolyzed whey permeate, and
the biomass yield and lipid yield plateaued at 9 g/L and
2.2 g/L, respectively (Chan et al. 2018).
Crude glycerol is a byproduct of biodiesel production,
with approximately 10 kg generated per 100 kg of bio-
diesel produced (Gao et al. 2016). To produce microbial
lipids, glycerol is utilized as a carbon source for several
yeast strains such as Rhodotorula, Lipomyces, Rho-
dosporidium, Cryptococcus, Trichosporonoides, and Yar-
rowia (Qin et al. 2017). R. toruloides has been extensively
studied due to its ability to produce microbial lipids when
cultured on glycerol. R. toruloides strain AS2.1389 dis-
played exceptional lipid production performance, with a
maximum yield of 26.7 g/L and a lipid content of 70% in a
5 L fermenter (Xu et al. 2012). The effect of impurities in
crude glycerol on the lipid production of Rhodosporidium
toruloides 32489 was investigated. Methyl oleate, NaCl,
and sodium oleate impurities boosted lipid production by
47.0%, 64.0%, and 68.0%, respectively, whereas methanol
lowered lipid generation by 17.7%. However, the inhibi-
tory effect of methanol was alleviated when mixed with
other impurities owing to their promoting effect (Gao et al.
2016).
Starch, an energy-storage polysaccharide formed by amy-
lopectin and amylose chain helix (Ledesma-Amaro et al.
2015), is one of the most abundant carbohydrates in nature
(Ge et al. 2018). Mortierella and Cryptococcus typically
use starch for microbial lipid synthesis because fungi can
produce starch hydrolysis enzymes (Zhang et al. 2016).
Environmental Science and Pollution Research (2023) 30:79654–79675
“Ex novo” lipid fermentation
In hydrophobic environments, certain strains of microor-
ganisms produce lipids via an ex novo lipid accumulation
pathway. This process involves the conversion of hydropho-
bic substrates to free fatty acids via microbial lipolytic pro-
cesses, followed by active transport within microbial cells
(Vyas et al. 2023). As microorganisms are only supplied
with hydrophobic substrates, this growth-oriented process
is independent of nitrogen-limiting conditions (Huang et al.
2017). Hydrophobic substrates can be used by oleaginous
yeasts, such as Rhodosporidium, Cryptococcus, Trichos-
poron, and Yarrowia (Patel and Matsakas 2019).
Hydrophobic feedstocks, such as free fatty acids from
industrial waste liquids, waste cooking oils (WCOs),
fatty acid byproducts or wastes, animal or vegetable oils,
n-alkanes, and volatile fatty acids (VFAs), can serve as
sources of lipids for oleaginous yeasts. Patel and Matsakas
(2019) evaluated the potential of Cryptococcus curvatus to
utilize waste cooking oils. Based on their findings, ex novo
lipid synthesis resulted in a significantly higher lipid con-
centration, cell dry weight, and lipid content than growth
on glucose. Additionally, VFAs derived from waste can be
utilized for biodiesel production using oleaginous yeasts,
such as Rhodosporidium toruloides, Cryptococcus curva-
tus, and Yarrowia lipolytica. Yarrowia lipolytica has been
found to effectively synthesize lipids using VFAs as a carbon
source, and the simultaneous biovalorization of VFAs and
glycerol or glucose resulted in a high lipid yield of 16.50 g/L
(Fontanille et al. 2012).
In the “ex novo” lipid accumulation pathway, only
0.25–0.35 g of microbial lipid can be produced for every
1.0 g of initial substrate fat consumed (Raut et al. 2022).
Additionally, after the conversion of extracellular lipids to
intracellular fat, a series of steps including breakage and
extraction are required, which increased production costs
(Mendoza-Lopez et al. 2017). Thus, it is impractical to per-
form fat substrate fermentation solely to produce another
intracellular fat. Gao et al. (2022) found that the combined
use of glucose and WCO as feedstock resulted in a higher
lipid production of 5.65 g/L, which was 32.0% more than
that achieved by the two pure groups. This result was due to
the fatty acid salts generated from WCO under alkaline con-
ditions, which improved lipid production as a surface-active
agent. However, the mixed group had a substantially lower
WCO usage rate, indicating that the production of microbial
lipids from a single WCO is not economical.
The direct production of biodiesel from WCO is more
economical (Karmakar et al. 2021). Two main methods are
used to produce biodiesel from WCO: esterification and
thermal treatment. Esterification involves the conversion
of triglycerides into methylesters using methanol, whereas
thermal treatment involves thermal cracking, catalysts, and
79659
hydrotreatment (Zhang et al. 2023). Thermochemical trans-
formations, such as pyrolysis, achieved by thermal pyrolysis
or catalytic cracking, are now widely employed. The selec-
tion of a suitable catalyst for this process is crucial to achieve
highly efficient biofuel production and resource separation
(Ahmed et al. 2022). The acid-catalyzed process utilizing
WCO has been found to be more technically viable and less
complicated than the alkali-catalyzed approach, thereby
presenting a competitive option for commercial biodiesel
production (Zhang et al. 2003).
In general, when grown on N-limited substrates such as
glucose or glycerol, strains of Yarrowia typically produce
limited quantities of lipids during the early growth stages,
with these lipids often being subjected to biodegradation
thereafter. Simultaneously, cells synthesize low-molecular-
weight compounds such as citric acid and polyols (Khot et al.
2020). However, lipid production by wild-type strains of this
species is generally inferior to that of wild-type strains of
Rhodotorula, Rhodosporidium, Lipomyces, and other typical
oleaginous yeasts (Chatterjee and Mohan 2020). For exam-
ple, under ideal cultivation conditions, Rhodosporidium
toruloides can accumulate lipids in excess of 70% (w/w) of
its biomass (Ma et al. 2021). The lipid contents of Lipomyces
and Rhodotorula were reported to be as high as 77.8% and
68.0%, respectively, whereas some species of Yarrowia (e.g.,
Yarrowia lipolytica) can only accumulate 38% of the lipid
content (Gujjala et al. 2019).
Feedstock and corresponding products
Research on microbial lipid from numerous raw materials is
mainly focused on biodiesel (Fig. 4), because the FA com-
positions of microbial lipid are comparable to vegetable oils
commonly used for the synthesis of first-generation biodiesel
(Santek et al. 2018). The utilization of microbial sources for
biodiesel production has been considered the most reliable
and appropriate strategy to meet the productive, technical,
and environmental demands (Sitepu et al. 2014). However,
the high cost of raw materials is a key factor that limits
the popularity and application of microbial lipid (Cho and
Fig. 4  Frequently studied feedstocks and the corresponding products
13
79660
Park 2018). For example, the cost of preparing biodiesel
from glucose and starch is over 20,000 yuan per tonne, with
raw material costs accounting for 60–85% of the total cost
(Shields-Menard et al. 2018). Therefore, the development of
low-cost and easily available raw materials is one of the hot
spots for research into products derived from microbial lipid.
In addition, it is worth noting that small amounts of
microbial lipid can also be very valuable. For example, 1 kg
of DHA is worth 700 RMB, while 1 ton of biodiesel is worth
6000 RMB (Gao et al. 2019). Therefore, with constant yield,
if the lipid produced contains more than 0.86% DHA, the
value of DHA in the lipid will be higher than that sold as
biodiesel. As a result, the valorization of low-cost raw mate-
rials towards the production of value-added PUFAs such as
GLA, EPA, ARA, and DHA is attracting increasing interest.
EPA, particularly ethyl esters, is commercialized for
hypertriglyceridemia treatments. Lignocellulosic biomass is
a frequently studied source that can be utilized as a low-cost
C source for PUFA synthesis, not only because lignocellu-
losic biomass is a renewable supply of sugars for the produc-
tion of value-added products but also because it is a plentiful
waste stream (Arevalo-Gallegos et al. 2017). Moreover, as
pyrolysis becomes a profitable method for depolymerizing
lignocellulose into assimilable C, the number of studies on
it grows dramatically (Palazzolo and Garcia-Perez 2022).
EPA could be produced by microalgae Nannochloropsis
and Crypthecodinium species with lignocellulose (Kar-
naouri et al. 2020; Palazzolo and Garcia-Perez 2022). Since
microalgae Nannochloropsis contain abundant long-chain
polyunsaturated fatty acids and are commonly used for EPA
production (Kumar et al. 2022). The use of wastewater for
EPA production has also been frequently investigated, with
studies indicating the possibility of using wastewater from
pesticides, pharmaceuticals, municipality sewage, petro-
leum, and the paper industry as a growth medium for Nan-
nochloropsis to produce EPA (Mitra et al. 2016; Polishchuk
et al. 2015). Euglena gracilis grown in aquaculture waste-
water showed the highest EPA content of 7.6 mg/g (Tos-
savainen et al. 2019).
DHA is essential for correct brain and visual development
in newborns (Palazzolo and Garcia-Perez 2022). Crypthe-
codinium cohnii is well-known for producing high amounts
of DHA-enriched lipids, with DHA content typically rang-
ing from 30 to 50%. To achieve efficient DHA production,
lignocellulosic hydrolysates are often used as feedstocks for
C. cohnii (Asimakopoulou et al. 2021; Karnaouri et al. 2020,
2021). Thraustochytrids are good heterotrophic microorgan-
isms for the commercial production of DHA (Patel et al.
2020), and Aurantiochytrium and Schizochytrium are com-
monly studied for wastewater treatment in combination with
DHA production because of their ability to accumulate large
quantities of DHA (Bagul and Annapure 2020; Chen et al.
2016b; Humaidah et al. 2020). The regulations governing
13
Environmental Science and Pollution Research (2023) 30:79654–79675
DHA production primarily focus on ensuring product safety,
particularly regarding environmental contaminants. National
governments have set maximum permitted levels of con-
taminants in food products to decrease the risk of dietary
exposure and safeguard consumers. In addition to safety
restrictions, some governments and organizations enforce
quality criteria. The European Union (EU), China, and the
USA are key markets for DHA products and typically lead in
setting regulations. Other national governments often follow
the regulations of these major consumer countries or adopt
international guidelines, such as those set by the FAO/WHO
CODEX Alimentarius (Auchterlonie and Bescoby 2021).
New products may require additional regulatory approval,
such as the three algal oil products that have acquired EU
regulatory clearance for both food and innovative food uses
(Winwood 2013). National regulations, such as those in
China, aim to control contaminants in food products. The
standards set by the China Administration for Quality Super-
vision Inspection and Quarantine and the China Food and
Drug Administration include both safety requirements and
quality specifications. The CODEX ALIMENTARIUS and
trade groups, such as the Global Organization DHA, pro-
mote Chinese standards that include safety regulations, qual-
ity criteria, and fair practices in international food trading
(Auchterlonie and Bescoby 2021).
Oleaginous microbes
Based on the bibliometric analysis, oleaginous microbes
have emerged as a significant area of research in the field
of microbial lipid production. Oleaginous microorganisms
are defined as microbes that can accumulate more than 20%
of their dry body mass as lipids, primarily in the form of
TAG and FA (Jin et al. 2015). The lipid contents of efficient
oleaginous microbes averagely reach 40% of their dry mass,
and even up to 70% of cell dry mass in some strains under
optimal conditions (Palazzolo and Garcia-Perez 2022). Ole-
aginous microbes predominantly comprise bacteria, yeast,
mold, and microalgae (Fig. 5). Among the several types of
most studied microorganisms, oleaginous yeast generally has
a higher biomass and lipid content.
Oleaginous bacteria
Oleaginous bacteria generally accumulate polyhydroxyal-
kanoates (PHAs) for energy storage, while TAGs, mainly
C16:0, C18:0, and C18:1, are synthesized by some strains
under N-limiting conditions, such as Mycobacterium, Nocar-
dia, Rhodococcus, and Streptomyces (Goswami et al. 2017).
Rhodococcus spp., particularly R. opacus and R. jostii, are
potential microbial cell factories for lipid generation and
have gained special interest due to their capacity to convert
Environmental Science and Pollution Research (2023) 30:79654–79675
Fig. 5  Comparison of the
production of microbial lipids
among different oleaginous
microbes
agro-industrial wastes into value-added lipids (Cho et al.
2020). Some other bacteria, such as cyanobacteria (Eun-
grasamee et al. 2019) and Methylomicrobium buryatense
(Demidenko et al. 2017), could produce FAs and deriva-
tives using carbon dioxide ­(CO2) and methane, respectively.
Bacteria usually generate less lipid per gram of biomass than
other oleaginous microbes (Bellou et al. 2016). Furthermore,
lipids adhere strongly to the bacterial membrane, making
them difficult to extract (Bao et al. 2021). Thus, the indus-
trial application of bacteria for lipid production is limited.
However, compared to other microbes, the genetic regulation
mechanism of oleaginous bacteria in FAs synthesis is more
thorough, so bacteria could be genetically manipulated to
enhance lipid production. For example, the titers of ethyl
esters FA and long-chain hydrocarbons produced by meta-
bolically engineered R. opacus were obviously improved
(Kim et al. 2019).
Oleaginous mold
Oleaginous molds include Mortierella, Aspergillus,
Dmsporangium, Cunnighamella, Mucor, Saprolegnia, and
Pichia (Donot et al. 2014) and can store lipids up to 80%
of their biomass (Pinzi et al. 2014). Mold-produced micro-
bial lipids are more unsaturated than yeast-produced lipids
(Papanikolaou and Aggelis 2011), and most molds have
been explored to produce special lipids rich in PUFAs,
such as DHA, EPA, γ-linolenic acid (GLA), and ARA,
which have attracted much medical and dietetical inter-
est. Several microbial lipids derived from molds are now
commercially available, including GLA from Mucor cir-
cinelloides and ARA from Mortierella alpina (Khot et al.
2020). However, mold lipid use for biodiesel synthesis is
still at the laboratory scale, and there are few publications
on the utilization of mold lipids for biodiesel production
79661
(Liang and Jiang 2013). Furthermore, unlike microalgae,
mold can grow in traditional bioreactors, lowering the cost
of lipid synthesis (Chintagunta et al. 2021).
Oleaginous microalgae
Microalgae grow autotrophically and can convert ­CO 2
into high-value products by photosynthesis in most cases
(Azarpour et al. 2022; Das et al. 2021). However, micro-
algae can also be cultivated under heterotrophic condi-
tions or mixed conditions with organic C amendment (Liu
et al. 2021). Compared to autotrophic microalgae, hetero-
trophic microalgae have shown a 900% increase in lipid
yield (Tomas-Pejo et al. 2021). Under heterotrophic con-
ditions, the microalgae could accumulate a high amount
of omega-3 fatty acids such as DHA and EPA, as well
as omega-6 fatty acids such as GLA and AA (Das et al.
2021; Karnaouri et al. 2020; Kumar et al. 2022). Further-
more, microalgae can grow in salt or seawater (Park et al.
2019). Most microalgae generate small amounts of TAGs;
however, when exposed to salicylic acid stress, Nanno-
chloropsis sp. achieved a high TAG yield of 475 mg/L
(Udayan et al. 2020). Among various microalgal species,
Chlorella vulgaris has been frequently used for TAG and
FA production due to its potential for breeding on a large
scale (Jeevan Kumar et al. 2020; Sakarika and Kornaros
2019). Microalgae exhibit several unique characteristics,
including high ­CO2 utilization efficiency, greater lipid pro-
ductivity than most commercially used plants, minimum
land requirement, and autotrophic mode of nutrition (Park
et al. 2019). However, low growth rates and high dewater-
ing costs are the major drawbacks of microalgal FAs and
TAGs production (Cho et al. 2020).
13
79662
Oleaginous yeast
Oleaginous yeasts are the most studied microbes owing to
their rapid growth rate, viral infection resistance, and capac-
ity to reach high cell densities (Kamineni and Shaw 2020).
The FA distribution in oleaginous yeast is relatively sim-
ple, with hexadecanoic acid and octadecanoic acid as the
main fatty acids, similar to many vegetable oils. The TAG
content is high in lipids produced by yeast, accounting for
90% of the storage of lipids (Donot et al. 2014). Among
oleaginous microorganisms, yeast shows the greatest genus
diversity (Palazzolo and Garcia-Perez 2022). Approximately
5% of the 1600 known yeast species are oleaginous (Garay
et al. 2016). The extensively studied lipid-producing yeasts
include Rhodotorula glutinis, Lipomyces Starkey, Rho-
dosporidium toruloides, Cryptococcus curvatus, and Yar-
rowia lipolytica (Ma et al. 2021). Y. lipolytica possesses an
evolutionarily advanced C-to-lipid flow, which is activated
during the stationary growth phase under N limited and
C surplus conditions (Takaku et al. 2020). R. toruloides
and L. starkeyi exhibit greater metabolic flexibility than Y.
lipolytica (Spagnuolo et al. 2019). Oleaginous yeasts have
significant benefits over other forms of oleaginous microor-
ganisms, including ease of culture and resistance to bacte-
rial contamination (Santamauro et al. 2014). In addition, as
oleaginous yeast can utilize different low-cost feedstocks,
they are promising alternative lipid sources (Karamerou and
Webb 2019).
Cultivation modes of oleaginous microbes
The culture mode can affect the yield and productivity of
microbial lipid (Karamerou and Webb 2019). The primary
culture modes may be categorized into batch, fed-batch,
and continuous based on the various nutrient amendment
Fig. 6  Comparison of several
common cultivation modes
utilized for lipid production
13
Environmental Science and Pollution Research (2023) 30:79654–79675
methods (Fig. 6). To obtain high cell density and product
yield, the fed-batch mode is preferred over other cultiva-
tion strategies (Chatterjee and Mohan 2020). Owing to the
different needs of oleaginous microbes at different stages,
two-stage fermentation or two-stage fed-batch cultivation
have received substantial attention in recent years (Boonyarit
et al. 2020; Polburee and Limtong 2020; Poontawee and
Limtong 2020).
Batch mode
Batch culture refers to adding all the reaction components
into the flask or bioreactor at the beginning. It is a funda-
mental and well-studied mode for lipid synthesis (Karam-
erou and Webb 2019). The batch mode in flasks is frequently
used for media optimization or exploring the lipid produc-
tion capacities of oleaginous strains (Chang et al. 2015;
Huang et al. 2016b; Tchakouteu et al. 2015a). When cultured
in a bioreactor, optimal fermentation conditions for lipid
synthesis were studied, including pH (Cavero-Olguin et al.
2021) and nutrient concentrations (Cho et al. 2017). The
results of batch cultivations are shown in Table 1. Because
a small amount of N was supplied in batch mode, the initial
concentration of nutrients in the medium limits the growth
of bacteria. Simply increasing the initial concentration of
nutrients may sometimes fail to improve the biomass and
lipid yield; on the contrary, it may have a toxic effect on the
strains (Christophe et al. 2012). Many researchers have real-
ized that it is difficult to improve the lipid content in batch
mode, so other culture modes have been studied and applied.
Fed‑batch mode
Fed-batch fermentation refers to adding substrates into
the reactor multiple times (Kumar et al. 2021; Zhao et al.
2022). One of the most frequent strategies for microbial
Environmental Science and Pollution Research (2023) 30:79654–79675 79663

Table 1  A summary of the yields produced during batch fermentation on different substrates
Strain Carbon source Scale DCW (g/L) Lipid content (%) Ref

Rhodosporidium toruloides Crude glycerol Shake flask 14.85 41.76 (Gao et al. 2016)
Rhodosporidium toruloides Glucose 3.5 L bioreactor 36.2 65.1 (Tchakouteu et al. 2017)
Rhodosporidium paludigenum Enzymatic corncob hydrolysate 3.0 L bioreactor 27.77 70 (Chaiyaso et al. 2019)
Rhodosporidium toruloides Glycerol 1 L bioreactor 17.7 42.13 (Lopes et al. 2020)
Rhodotorula graminis Corn stover hydrolysate 1 L bioreactor 48 34 (Galafassi et al. 2012)
Rhodotorula glutinis Pure glycerol 2 L bioreactor 5.3 33 (Karamerou et al. 2016)
Lipomyces starkeyi Crude glycerol Shake flask 16.2 54.3 (Tchakouteu et al. 2015b)
Lipomyces starkeyi Hemicellulose hydrolysate 1.3 L bioreactor 9.6 26.1 (Xavier et al. 2017)
Cryptococcus curvatus Waste spent yeast Shake flask 50.4 37.7 (Ryu et al. 2013)

lipid synthesis in this process is the intermittent supply Continuous mode

of glucose without the removal of the fermentation broth
(Chang et al. 2013). Fed-batch cultivation is an effective
mode for achieving higher cell densities, lipid content,
and lipid yield (Anschau et al. 2014; Chen et al. 2018).
Results obtained from fed-batch cultivation modes are
displayed in Table 2. Compared to batch mode, fed-batch
culture minimizes the inhibition of high concentrations of
substrate and end-products on the growth and metabolism
of the oleaginous microbes (Fei et al. 2016). In addition,
the operation of the fed-batch mode is as easy and reli-
able as batch mode (Salehmin et al. 2013). However, the
performance of the fed-batch mode is strongly reliant on
the feeding strategy used during culture (Chen et al. 2018;
Kumar et al. 2021). Due to their relatively simple and low
cost, simple indirect feedback control strategies combin-
ing substrate feeding with pH or dissolved oxygen (DO)
determination have been employed for many years. Nev-
ertheless, the sluggish response of cells to the changes
in pH or DO caused by substrate usage could result in
over- or under-feeding, which might affect microbial lipid
production (Fei et al. 2016).
Continuous mode involves continuously adding fresh sub-
strates to a well-stirred culture at a certain speed, and prod-
ucts and cells are withdrawn at the same speed to maintain a
stable C/N ratio in the fermentation system (Christophe et al.
2012). Continuous culture outperforms batch mode in terms
of lipid productivity and economic viability since it enables
the collection of products during the fermentation process
(Karamerou and Webb 2019). In addition, the continuous
feeding supply prevents the cells from being inhibited by
a large amount of substrate. Continuous mode allows for
parameter isolation and exploration of its impact on lipid
synthesis while maintaining the other parameters constant,
such as cell concentration, growth, and substrate absorp-
tion (Anschau et al. 2014). In continuous cultivation, the
main factor that affects lipid productivity is the dilution rate
(Karamerou and Webb 2019). Since cell proliferation and
product synthesis could be inhibited under high concentra-
tions of such wastes. In a broader context, continuous feed-
ing at low dilution rates is more advantageous for the utili-
zation of low-cost wastes rich in C, such as lignocellulosic
hydrolysates and crude glycerol, because these wastes often
contain a large number of toxic compounds (Gorte et al.
2020). The lipid production during continuous cultivations

Table 2  Summary of lipid productions obtained via fed-batch cultivation

Strain Carbon source Scale DCW (g/L) Lipid content (%) Ref

Rhodosporidium toruloides Glucose 15 L bioreactor 106.5 67.5 (Li et al. 2007)

Rhodosporidium toruloides Cornstalk hydrolysate 15 L bioreactor 78.7 61.8 (Zhao et al. 2011)
Rhodosporidium paludigenum Enzymatic corncob hydrolysate 3.0 L bioreactor 36.06 70 (Chaiyaso et al. 2019)
Cryptococcus sp. Glucose and corncob hydrolysate Shake flask 10.8 61.3 (Chang et al. 2013)
Lipomyces starkeyi Glucose and xylose 1 L bioreactor 86.4 36.2 (Anschau et al. 2014)
Rhodotorula glutinis Crude glycerol 50 L bioreactor 39.2 43.3 (Yen et al. 2015)
Trichosporon oleaginosus Crude glycerol 15 L bioreactor 21.87 49.89 (Chen et al. 2017)
Cutaneotrichosporon oleagino- Alkaline organosolv-pretreated Shake flask 50.7 61.7 (Gong et al. 2020)
sum corn stover

13
79664 Environmental Science and Pollution Research (2023) 30:79654–79675

at various dilution rates is summarized in Table 3. The con-
tinuous mode may be superior to the batch mode because of
its long-term operation and consistent biomass. Long-term
fermentation, on the other hand, may result in blockages due
to biofilm development or medium sedimentation, as well
as contamination and changes in cell viability, which may
reduce overall yield (Karamerou and Webb 2019).
Two‑stage fermentation
Microbial lipid production consists of two stages, in which
the biomass first grows, and then lipid accumulation occurs.
The cell proliferation phase requires a rich medium, whereas
the lipid accumulation requires only a carbon source (Xu
et al. 2017). The difference in nutritional demand between
biomass growth and lipid yield presents a dilemma (Fei
et al. 2015; Xu et al. 2017). Two-stage cultivation is used
to separate the cell proliferation and lipid accumulation
phases, thereby creating an environment suitable for biomass
growth and lipid accumulation. This mode allows oleaginous
microbes to increase biomass under suitable conditions and
then transfer to another system for lipid accumulation, which
has been demonstrated to improve lipid production (Zhang
et al. 2021b). Two-stage fermentation can be implemented
in both fed-batch and continuous modes and allows easy
adjustment of the two stages by modifying the nutrient input
(Karamerou and Webb 2019). Boonyarit et al. (2020) used
a two-stage batch mode strategy with sugarcane top and
crude glycerol as feedstocks, R. fluvialis used sugarcane top
hydrolysate as a substrate for cell proliferation at 200 r/min
and 28 °C, and crude glycerol solution was utilized for lipid
synthesis in the second stage. A high lipid concentration and
lipid content of 15.85 g/L and 73.04%, respectively, were
observed (Table 4). Two-stage fermentation is not limited
to nutrients; other factors, such as pH (Zhang et al. 2019a),
C/N ratio (Fu et al. 2018), DO (Zhang et al. 2019b), and tem-
perature (Polburee et al. 2016), could be adjusted to ensure
that the culture conditions are more targeted for both phases.
Zhang et al. (2019a) developed a two-stage fed-batch pH
regulation strategy using acetate as a substrate by R. glutinis.
As a result, a promoted biomass of 67.2 g/L at pH 6.6 and
an enhanced lipid titer of 29.3 g/L at pH 7.0 were achieved.
The biomass and lipid titer increased 15.6-fold and 19.1-
fold, respectively, compared to those in the one-stage batch
mode. Optimizing the DO concentration during the growth
and lipogenesis phases can enhance the lipid content of T.

Table 3  The effect of the dilution rates on the lipid production under continuous cultivations
Strain Carbon source Dilution rate DCW (g/L) Lipid con- Ref
­(h−1) tent (%)

Schizochytrium sp. Glucose and glutamate 0.02 7.7 31 (Ganuza and Izquierdo 2007)
Rhodosporidium toruloides Glucose 0.02 8.67 8.66 (Shen et al. 2013)
Cryptococcus curvatus Acetate 0.04 1.34 56.7 (Gong et al. 2015)
Lipomyces starkeyi Glucose and xylose 0.06 10 43.3 (Anschau et al. 2014)
Yarrowia lipolytica Crude glycerol 0.03 8.1 43.3 (Papanikolaou and Aggelis 2002)
Yarrowia lipolytica Glycerol 0.01 59.8 31 (Rakicka et al. 2015)
Rhodotorula glutinis Glucose 0.02 27 35.2 (Shen et al. 2020)

Table 4  Overview of lipid production on two-stage cultivations

Strain Operation DCW (g/L) Lipid content (%) Ref

Rhodosporidium toruloides Cultivation in glucose medium followed by resuspen- 6.75 50.1 (Huang et al. 2016b)
sion in acetic acid
Lipomyces starkeyi Growth in a 15 L bioreactor using glucose-rich medium 104.6 64.9 (Lin et al. 2011)
and resuspension in a 7 L bioreactor using glucose
media
Cryptococcus curvatus Cultivation in N-acetylglucosamine media and resus- 16.8 54.2 (Tang et al. 2018)
pension to N-glucosamine solution
Rhodosporidiobolus fluvialis Cultivation in sugarcane top hydrolysate medium fol- 21.07 73.04 (Boonyarit et al. 2020)
lowed by resuspension in crude glycerol
Rhodosporidium toruloides Cultivation in food waste hydrolysate with pH of 4.0 13.86 50 (Gao et al. 2019)
and resuspension with pH of 11.0 in shake flask
Yarrowia lipolytica 60 h of growth in glycerol medium followed by the 41.02 34.59 (Fontanille et al. 2012)
addition of VFAs in a 7 L bioreactor

13
Environmental Science and Pollution Research (2023) 30:79654–79675 79665

oleaginosus while reducing energy consumption. Zhang
et al. (2019b) found that maintaining a high DO concentra-
tion during the growth stage and a low DO concentration
during the lipogenesis phase allowed the supply of only the
necessary oxygen for the yeast.
Our group developed a two-stage fermentation mode: ex
situ and in situ. As we found that lactic acid can promote
cell proliferation but inhibit lipid accumulation, a two-stage
fermentation was developed using R. toruloides in differ-
ent systems with high and low lactic acid concentrations.
After 4 days of cell proliferation in 100 mL of saccharified
food waste liquid (lactic acid concentration of 8.3 g/L), the
bacteria were transferred to 100 mL of saccharified liquid
(lactic acid concentration of 0.3 g/L) prepared from fresh
food waste for 6 days for lipid accumulation. Compared to
traditional one-stage fermentation, this ex situ two-stage fer-
mentation resulted in a total lipid concentration of 9.19 g/L
and a lipid yield of 0.204 g/g total sugar, which increased by
44.27% and 60.63%, respectively (Ma et al. 2019). In addi-
tion, as an acidic environment (pH 4.0) can promote the cell
proliferation of R. toruloides and an alkaline environment
(pH 11.0) can promote lipid accumulation, an in situ two-
stage fermentation mode based on pH in the same medium
was developed; that is, after adjusting the pH of the hydro-
lysate to 4.0, sterilization and inoculation for acid stage fer-
mentation were conducted for 1 day. The pH value was then
adjusted to 11.0, and the yeast was fermented in an alkaline
stage for 5 days, in which the maximum lipid production was
6.94 g/L, which was 84.6% higher than that in the traditional
single-stage fermentation (Gao et al. 2019). Alkaline condi-
tions increase the degree of WCO hydrolysis in food waste
hydrolysis solutions, and the generated fatty acid salts act
as surfactants that promote the utilization of the substrate
by oleaginous yeast (Gao et al. 2022). In conclusion, the ex
situ two-stage fermentation mode can tolerate greater lac-
tic acid concentrations in the substrate, whereas the in situ
fermentation mode enables greater lipid production and has
the potential for practical use.
Metabolic engineering strategies and lipid
production
The yield of microbial lipid production is closely linked
to the flow of metabolic pathways in oleaginous microor-
ganisms. The use of glucose by oleaginous yeasts has been
extensively studied with respect to microbial lipid synthesis
(Fig. 7). A condition in which there is an excess of C and
a deficiency of N can induce lipid synthesis (McNeil and
Stuart 2018). The depletion of N results in increased AMP
deaminase activity, leading to the breakdown of adeno-
sine monophosphate (AMP) into inosine 5′-monophos-
phate (IMP) and ammonia, and ammonia is an emergency
response to N depletion. Low levels of AMP could dimin-
ish the activity of isocitrate dehydrogenase (IDH), a unique
effect found only in oleaginous species (Kosa and Ragauskas
2011), resulting in citrate accumulation in the mitochon-
dria (Zhang et al. 2022). The ATP-citrate lyase (ACL) will
cleave the accumulated citrate into acetyl-CoA and oxaloac-
etate (OAA) in the cytosol after it is exported by the cit-
rate/malate translocase (CMT) (Chattopadhyay et al. 2021).
Subsequently, OAA is converted to malate, which is further
converted to pyruvate in the presence of a malic enzyme
(ME). Acetyl-CoA carboxylase (ACC) converts a part of
acetyl-CoA into malonyl-CoA (Kamineni and Shaw 2020).
Then fatty acid synthase (FAS) involves both acetyl-CoA
and malonyl-CoA in the synthesis of acyl-CoA, which is

Fig. 7  Simplified overview of

lipid synthesis and the regula-
tory pathways of oleaginous
yeast. ER, endoplasmic reticu-
lum; MT, mitochondria; ACL,
ATP-citrate lyase; MDH, malate
dehydrogenase; ME, malic
enzyme; ACC, acetyl-CoA
carboxylase; FAS, fatty acyl
synthetase; M-ACP, malonyl
acyl-carrier protein; G3P,
glycerol-3-phosphate; GPAT,
Acyl-CoA: glycerol-sn-3-phos-
phate acyltransferase; PA,
phosphatidic acid; DAG, diacyl-
glycerol; DGAT, diacylglycerol
acyltransferase; TAG, triacylg-
lycerol; PUFA, polyunsaturated
fatty acid

13
79666 Environmental Science and Pollution Research (2023) 30:79654–79675

then incorporated into the Kennedy pathway to form triacyl-
glycerol (TAG) in the endoplasmic reticulum (Zhang et al.
2022).
Metabolic engineering strategies have been widely
employed in yeast and microalgae to reconfigure their
internal metabolic networks and enhance lipid produc-
tion (Jakhwal et al. 2022; Kamineni and Shaw 2020; Qiao
et al. 2015). These strategies include overexpressing genes
related to lipid synthesis and knocking out competing
pathways that flow to other byproducts (Table 5). Genes
involved in FA and TAG syntheses, such as ACC1, ME,
DGAT1, and DGA1, were generally overexpressed. Also,
genes that participated in the competing pathway, such
as ADP-glucose pyrophosphorylase (AGPase), UDP-glu-
cose pyrophosphorylase gene (UDPase), and lipase, were
knocked out or knocked down (Kang et al. 2022).

Table 5  Overviews of metabolic engineering approaches to enhance lipid synthesis in oleaginous microbes
Gene Approach Strains Phenotypic changes REF

FA and TAG biosynthesis

ACC​ Overexpression of PtACC2 Phaeodactylum tricornutum 1.77-fold increase in lipid (Li et al. 2018)
content
ACC​ Overexpression of ACC​ Schizochytrium sp. 24% increase in DHA (Han et al. 2020)
ACC​ Heterologous expression of Saccharomyces cerevisiae 24% increase in lipid (Wang et al. 2016)
LsACC1 content
ME Heterologous expression Rhodotorula mucilaginosa 1.18-fold increase in lipid (Bandhu et al. 2019)
of ME production
ME Overexpression of NsME1 Nannochloropsis salina 53% increase in FA methyl (Jeon et al. 2021)
ester yield
ME Overexpression of ME Chlorella protothecoides 2.8-fold increase in lipid (Yan et al. 2019)
accumulation
FAS Overexpression of FAS1 and Saccharomyces cerevisiae 53% increase in lipid (Runguphan and Keasling
FAS2 content 2014)
FAS Overexpression of FAS1 Yarrowia lipolytica 33% increase in lipid titer (Silverman et al. 2016)
GPAT Overexpression of AGPAT1 Phaeodactylum tricornutum 1.81-fold increase in TAG (Balamurugan et al. 2017)
content
GPAT Overexpression of GPAT2 Phaeodactylum tricornutum 2.9-fold increase in TAG (Wang et al. 2020c)
content
GPAT Heterologous expression of Saccharomyces cerevisiae 1.3-fold in lipid content (Wei et al. 2017)
GPAT
DGAT​ Heterologous expression of Scenedesmus obliquus 2-fold increase in lipid (Chen et al. 2016a)
DGTT1 content
DGAT​ Overexpression of DGAT1 Nannochloropsis oceanica 2.4-fold and 47% increase in (Wei et al. 2017)
TAG content and yield
DGAT​ Overexpression of DGAT1 Phaeodactylum tricornutum 2.3-fold increase in TAG (Wang et al. 2018)
and LPAT1 content
DGAT​ Overexpression of DGA1 Yarrowia lipolytica 4-fold increase in lipid (Tai and Stephanopoulos
production 2013)
DGAT​ Overexpression of DGA1 Rhodosporidiobolus 2.5-fold increase in lipid (Polburee et al. 2018)
fluvialis content
DGAT​ Heterologous expression of Candida phangngensis 32% increase in lipid titer (Quarterman et al. 2018)
DGA1
Competitive pathways
AGPase Knockout Chlamydomonas 10-fold increase in TAG​ (Li et al. 2010)
UGPase Knockout Phaeodactylum tricornutum 45-fold increase in TAG​ (Daboussi et al. 2014)
YlGSY1 Knockout Yarrowia lipolytica 60% increase in TAG​ (Bhutada et al. 2017)
YlTGL3 and YlTGL4 Knockout Yarrowia lipolytica 10% increase in TAG (Dulermo et al. 2013)
content
CrACX2 Knockout Chlamydomonas 20% increase in TAG (Kong et al. 2017)
content

13
Environmental Science and Pollution Research (2023) 30:79654–79675
Enhancing FA and TAG synthesis via overexpressing
of lipid synthesis‑related genes
When it comes to lipid synthesis, acetyl-CoA carboxy-
lase (ACC) and diacylglyceride acyl-transferases (DGAT)
are two enzymes that have received considerable attention
(Tomas-Pejo et al. 2021). ACC catalyzes the first rate-limit-
ing step in FA production and has therefore been primarily
targeted (Park et al. 2019). Overexpressions of the lipid path-
way gene in oleaginous yeast, ACC1, have been reported,
but relatively smaller improvements have been achieved (Tai
and Stephanopoulos 2013; Wang et al. 2016). In contrast,
overexpression of endogenous ACC2 in microalgae P. tri-
cornutum resulted in 1.77-fold increases in the neutral lipid
content (Li et al. 2018).
Oleaginous microbes are capable of synthesizing large
quantities of TAG under N-restricted conditions due to their
high DGAT activity (Kosa and Ragauskas 2011). Therefore,
multiple gene overexpression studies indicate that DGAT,
responsible for the final step of TAG synthesis, has a promi-
nent influence on productivity in oleaginous yeast (Blazeck
et al. 2014; Kamineni and Shaw 2020; Tai and Stephanop-
oulos 2013). DGA1 and DGA2 are two isozymes found in
oleaginous yeast, and overexpression of either has improved
cell-specific lipid productivity (Kamineni and Shaw 2020).
The overexpression of either DGA1 or DGA2 led to an
increase in lipid content of up to 33.8% and 39%, respec-
tively (Chattopadhyay et al. 2021). DGAT overexpression
is also a commonly attempted approach in microalgae, and
lipid production has been found to increase in most cases
(Iwai et al. 2014; Li et al. 2016). Microalgae have four types
of DGAT: DGAT1, DGAT2, DGAT3, and wax ester synthase/
acyl-coenzyme A (acyl-CoA): diacylglycerol acyltransferase
(WS/DGAT​) (Kang et al. 2022). Overexpression of DGAT1
in Nannochloropsis oceanica increased TAG content and
yield by 2.4-fold and 47%, respectively (Wei et al. 2017).
The WS/DGAT​overexpression in Phaeodactylum tricornu-
tum resulted in a 40% rise in TAG content (Cui et al. 2018).
Blocking the contribution of competitive pathways
Another effective strategy for enhancing lipid production is
to block competing metabolic pathways. Various gene edit-
ing techniques, such as meganucleases and transcription
activator-like effector nucleases (TALENs), interfering, and
antisense RNA methods (Levering et al. 2015), have been
employed to knockdown and knockout genes responsible
for byproduct production. These genes are involved in low-
molecular metabolites secretion (e.g., citric acid, polyols)
(Steiger et al. 2019), polysaccharide or PHA accumulation
(Bhutada et al. 2017), and the lipid degradation pathway
(Shin et al. 2019).
79667
Blocking enzymes involved in polysaccharide synthe-
sis can favor lipid production in various microorganisms
(Dourou et al. 2018). For instance, Bhutada et al. (2017)
deleted the ylGSY1 gene, which encodes glycogen synthase
in Y. lipolytica, and achieved an up to 60% increase in TAG
accumulation. In microalgae, blocking starch synthesis and
storage could be a more effective method to enhance TAG
accumulation than direct engineering of the lipid biosynthe-
sis pathway (Dourou et al. 2018). Several microalgal strains
utilize C to produce starch as the main storage metabolite
(Park et al. 2019). Inactivating AGPase in a Chlamydomonas
starchless mutant resulted in a tenfold rise in TAG (Li et al.
2010). Disruption of the UDPase in Phaeodactylum tricor-
nutum resulted in a 45-fold increase in TAG accumulation
(Daboussi et al. 2014).
Inhibiting β-oxidation is another potential strategy that
focuses on TAG mobilization and oxidation pathways
(Probst et al. 2016). As β-oxidation occurs in peroxisomes,
genes involved in peroxisome biogenesis, such as PEX3,
PEX10, and PEX11 in Y. lipolytica, have been deleted to
prevent TAG degradation (Lazar et al. 2018; Wang et al.
2020a). TAG mobilization by TAG lipases (TGLs) is the
initial step in the breakdown process; therefore, the down-
regulation of TGLs could inhibit TAG mobilization for
β-oxidation. Deletion of either or both YlTGL3 and YlTGL4
in Y. lipolytica resulted in an increase of 10% in TAG content
(Dulermo et al. 2013). Under N-limited circumstances, the
mutation of the CrACX2 gene, which encodes a member of
the acyl-CoA oxidase/dehydrogenase superfamily, led to a
20% rise in lipid contents in Chlamydomonas (Kong et al.
2017).
Environmental implications and future
directions
Several environmental issues have arisen as a result of
the use of fossil fuels, such as global warming caused by
greenhouse gas emissions produced during the combustion
of fossil fuels to generate electricity (Saha and Mukhopad-
hyay 2021). The swift rise of renewable energy sources is
in line with the concept of “more energy, less carbon emis-
sions”. Microbial lipid has been identified as an attractive
possibility for biochemical production. Biodiesel produced
from microbial lipids presents a number of benefits over
traditional petroleum-based diesel, such as being a safer
and cleaner alternative for the environment (Leong et al.
2018). A recent study indicated that converting global food
waste into biodiesel could potentially replace around 27.9
million tonnes of mineral diesel, leading to a reduction
of roughly 0.31 million tonnes of C ­ O 2 emissions (Gao
et al. 2022). Also, microbial lipid can be generated by
agro-industrial wastes, and part of it solves the problem
13
79668
of waste disposal (Cho and Park 2018). For example, the
usage of WCO to produce biodiesel has become a criti-
cal development direction, where biodiesel production
from WCO not only replaces petroleum-based diesel and
reduces environmental pollution but also improves the
industrial added value of waste oil, ultimately achieving
waste-to-energy goals (Gao et al. 2022). Additionally, lipid
fermentation wastewater can be used in various applica-
tions, such as bacterial cellulose production (Huang et al.
2016a) and phosphorus recovery (Wu et al. 2023). The
treatment of lipid fermentation wastewater could not pos-
sibly be another issue that increases the sustainability of
microbial production methods.
Large-scale microbial lipid production has been exten-
sively studied in the past, but the high costs associated
with commercial application remain a major challenge
(Chaturvedi et al. 2019). On the other hand, microbial con-
version processes have low product concentrations, which
increase downstream processing costs (Rodionova et al.
2022). To address these challenges, several approaches
need to be considered. First, it is crucial to further explore
low-cost, substantial, and sustainable feedstocks for lipid
synthesis. Waste streams for lipid production need to be
identified and adopted, such as lignocellulosic biomass,
which is the most abundant for energy recovery. However,
lignin is difficult to degrade. The future studies should
focus on the structural characterization of lignocellulosic
waste, mechanisms of degradation, and process develop-
ment for valorization. The pretreatment of raw substrates
is also necessary to enhance the efficiency of the process
(Qin et al. 2017). Second, selecting or encoding new
strains will definitively improve the lipid generation from
wastes. Although several key genes have been identified
through genetic engineering and the effects of these genes
have been studied independently, future research should
explore theories that explain how two or more genes inter-
act in the process. Furthermore, a systems biology technol-
ogy based on omics and computational models will aid in
the design and reconstruction of superior strains for lipid
production enhancement. Third, the fermentation process
is quite important to the total cost. Developing two-stage
fermentation modes might promote high lipid production
while supplying nutrients at the appropriate fermentation
phase. Additionally, studying the kinetics of the system
allows for a better understanding of the fluxes and interac-
tions between important factors, enabling the prediction of
different conditions (Karamerou and Webb 2019). Eventu-
ally, in the case of lipid-based products, the co-production
of high value-added products is required for economic via-
bility. More attention should be paid in future research to
the regulation of the C flow in the co-production process,
which will aid in achieving a maximum theoretical yield
of multiple products.
13
Environmental Science and Pollution Research (2023) 30:79654–79675
Conclusion
Microbial lipids, as renewable energy sources, may be a
promising approach to tackle the problems of fossil fuel
depletion, environmental contamination, and climate
change. However, large-scale microbial lipid development
is still limited owing to high processing costs and low yields.
The main opportunity is to study feedstock supply and meta-
bolic modifications to produce high-value-added products.
However, to actualize this progress, low-cost, substantial,
and sustainable raw materials must be explored. Highly
effective oleaginous strains must be selected or encoded,
and the involvement of two or more genes in lipid synthesis
must be clarified. In a single study, focusing on a particular
feedstock and examining its fermentation using a group of
oleaginous bacteria as both novel candidates and controls
were preferred. Simultaneously, exploring and stabilizing
the synthesis route of high-value-added products could
expand the commercial applications of microbial lipids.
Author contribution All authors contributed to the study’s concep-
tion and design. Material preparation, data collection, and analysis
were performed by Haishu Sun, Zhen Gao, and Lirong Zhang. Review
and editing were performed by Xiaona Wang, Ming Gao, and Qunhui
Wang. Supervision was performed by Xiaona Wang and Qunhui Wang.
The first draft of the manuscript was written by Haishu Sun, and all
authors commented on previous versions of the manuscript. All authors
read and approved the final manuscript.
Funding This work was supported by Tianjin Education Commission
Scientific Research Planning Project [grant number 2021KJ064] and
Guangdong Basic and Applied Basic Research Foundation [grant num-
ber 2022A1515110943].
Data availability Not applicable
Declarations
Ethics approval and consent to participate Not applicable
Consent for publication Not applicable
Competing interests The authors declare no competing interests.
References
Ahmed HA, Altalhi AA, Elbanna SA, El-Saied HA, Farag AA, Negm
NA, Mohamed EA (2022) Effect of reaction parameters on cata-
lytic pyrolysis of waste cooking oil for production of sustainable
biodiesel and biojet by functionalized montmorillonite/chitosan
nanocomposites. ACS Omega 7:4585–4594. https://​doi.​org/​10.​
1021/​acsom​ega.​1c065​18
Anschau A, Xavier MC, Hernalsteens S, Franco TT (2014) Effect of
feeding strategies on lipid production by Lipomyces starkeyi.
Bioresour Technol 157:214–222. https://​doi.​org/​10.​1016/j.​biort​
ech.​2014.​01.​104
Environmental Science and Pollution Research (2023) 30:79654–79675
Arevalo-Gallegos A, Ahmad Z, Asgher M, Parra-Saldivar R, Iqbal
HM (2017) Lignocellulose: a sustainable material to produce
value-added products with a zero waste approach—a review. Int
J Biol Macromol 99:308–318. https://d​ oi.o​ rg/1​ 0.1​ 016/j.i​ jbiom ​ ac.​
2017.​02.​097
Arous F, Jaouani A, Mechichi T (2019) Oleaginous microorganisms for
simultaneous biodiesel production and wastewater treatment: a
review. Microbial Wastewater Treatment. Elsevier, pp 153–174
Asimakopoulou G, Karnaouri A, Staikos S, Stefanidis SD, Kalogiannis
KG, Lappas AA, Topakas E (2021) Production of omega-3 fatty
acids from the microalga crypthecodinium cohnii by utilizing
both pentose and hexose sugars from agricultural residues. Fer-
mentation-Basel 7. https://d​ oi.o​ rg/1​ 0.3​ 390/f​ ermen​ tatio​ n7040​ 219
Auchterlonie NA, Bescoby GH (2021) Global market for the long-
chain omega-3 fatty acids EPA and DHA and their regulation,
Omega-3 Delivery Systems. Elsevier, pp 79–106
Azarpour A, Zendehboudi S, Mohammadzadeh O, Rajabzadeh AR,
Chatzis I (2022) A review on microalgal biomass and biodiesel
production through Co-cultivation strategy. Energy Convers
Manage 267:115757. https://​doi.​org/​10.​1016/j.​encon​man.​2022.​
115757
Bagul VP, Annapure US (2020) Effect of sequential recycling of spent
media wastewater on docosahexaenoic acid production by newly
isolated strain Aurantiochytrium sp . ICTFD5. Bioresour Technol
306. https://​doi.​org/​10.​1016/j.​biort​ech.​2020.​123153
Balamurugan S, Wang X, Wang H, An C, Li H, Li D, Yang W, Liu J,
Li H (2017) Occurrence of plastidial triacylglycerol synthesis
and the potential regulatory role of AGPAT in the model dia-
tom Phaeodactylum tricornutum. Biotechnol Biofuels 10:1–14.
https://​doi.​org/​10.​1186/​s13068-​017-​0786-0
Bandhu S, Bansal N, Dasgupta D, Junghare V, Sidana A, Kalyan G,
Hazra S, Ghosh D (2019) Overproduction of single cell oil from
xylose rich sugarcane bagasse hydrolysate by an engineered
oleaginous yeast Rhodotorula mucilaginosa IIPL32. Fuel
254:115653. https://​doi.​org/​10.​1016/j.​fuel.​2019.​115653
Bao W, Li Z, Wang X, Gao R, Zhou X, Cheng S, Men Y, Zheng L
(2021) Approaches to improve the lipid synthesis of oleaginous
yeast Yarrowia lipolytica: a review. Renew Sust Energ Rev 149.
https://​doi.​org/​10.​1016/j.​rser.​2021.​111386
Bellou S, Triantaphyllidou I-E, Aggeli D, Elazzazy AM, Baeshen
MN, Aggelis G (2016) Microbial oils as food additives: recent
approaches for improving microbial oil production and its poly-
unsaturated fatty acid content. Curr Opin Plant Biol 37:24–35.
https://​doi.​org/​10.​1016/j.​copbio.​2015.​09.​005
Bhutada G, Kavšček M, Ledesma-Amaro R, Thomas S, Rechberger
GN, Nicaud J-M, Natter K (2017) Sugar versus fat: elimination
of glycogen storage improves lipid accumulation in Yarrowia
lipolytica. FEMS Yeast Res 17:fox020. https://​doi.​org/​10.​1093/​
femsyr/​fox020
Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, Otoupal P, Alper
HS (2014) Harnessing Yarrowia lipolytica lipogenesis to create a
platform for lipid and biofuel production. Nat Commun 5. https://​
doi.​org/​10.​1038/​ncomm​s4131
Boonyarit J, Polburee P, Khaenda B, Zhao ZBK, Limtong S (2020)
Lipid production from sugarcane top hydrolysate and crude
glycerol with Rhodosporidiobolus fluvialis using a two-stage
batch-cultivation strategy with separate optimization of each
stage. Microorganisms 8. https://​doi.​org/​10.​3390/​micro​organ​
isms8​030453
Brandenburg J, Blomqvist J, Shapaval V, Kohler A, Sampels S,
Sandgren M, Passoth V (2021) Oleaginous yeasts respond
differently to carbon sources present in lignocellulose hydro-
lysate. Biotechnol Biofuels 14:124. https://​doi.​org/​10.​1186/​
s13068-​021-​01974-2
Cai D, Dong Z, Wang Y, Chen C, Li P, Qin P, Wang Z, Tan T (2016)
Biorefinery of corn cob for microbial lipid and bio-ethanol
79669
production: an environmental friendly process. Bioresour
Technol 211:677–684. https://​doi.​org/​10.​1016/j.​biort​ech.​2016.​
03.​159
Carmona-Cabello M, Garcia IL, Papadaki A, Tsouko E, Koutinas
A, Dorado MP (2021) Biodiesel production using microbial
lipids derived from food waste discarded by catering services.
Bioresour Technol 323. https://​doi.​org/​10.​1016/j.​biort​ech.​2020.​
124597
Carota E, Crognale S, D’Annibale A, Gallo AM, Stazi SR, Petruc-
cioli M (2017) A sustainable use of Ricotta Cheese Whey for
microbial biodiesel production. Sci Total Environ 584:554–560.
https://​doi.​org/​10.​1016/j.​scito​tenv.​2017.​01.​068
Cavero-Olguin VH, Rahimpour F, Dishisha T, Alvarez-Aliaga MT,
Hatti-Kaul R (2021) Propionic acid production from glycerol
in immobilized cell bioreactor using an acid-tolerant strain of
Propionibacterium acidipropionici obtained by adaptive evolu-
tion. Process Biochem 110:223–230. https://​doi.​org/​10.​1016/j.​
procb​io.​2021.​08.​005
Chaiyaso T, Manowattana A, Techapun C, Watanabe M (2019) Effi-
cient bioconversion of enzymatic corncob hydrolysate into bio-
mass and lipids by oleaginous yeast Rhodosporidium paludige-
num KM281510. Prep Biochem Biotech 49:545–556. https://d​ oi.​
org/​10.​1080/​10826​068.​2019.​15919​85
Chan LG, Cohen JL, Ozturk G, Hennebelle M, Taha AY, de Moura
Bell JMLN (2018) Bioconversion of cheese whey permeate into
fungal oil by Mucor circinelloides. J Biol Eng 12:1–14. https://​
doi.​org/​10.​1186/​s13036-​018-​0116-5
Chang Y, Chang K, Hsu C, Chuang L, Chen C, Huang F, Jang H (2013)
A comparative study on batch and fed-batch cultures of oleagi-
nous yeast Cryptococcus sp in glucose-based media and corn-
cob hydrolysate for microbial oil production. Fuel 105:711–717.
https://​doi.​org/​10.​1016/j.​fuel.​2012.​10.​033
Chang Y, Chang K, Lee C, Hsu C, Huang C, Jang H (2015) Micro-
bial lipid production by oleaginous yeast Cryptococcus sp. in
the batch cultures using corncob hydrolysate as carbon source.
Biomass Bioenerg 72:95–103. https://​doi.​org/​10.​1016/j.​biomb​
ioe.​2014.​11.​012
Chatterjee S, Mohan SV (2020) Yeast fermentation towards biodiesel:
maximizing resource recovery by integrating with biohydro-
gen production in biorefinery framework. Biomass Bioenerg
142:105747. https://​doi.​org/​10.​1016/j.​biomb​ioe.​2020.​105747
Chattopadhyay A, Mitra M, Maiti MK (2021) Recent advances in lipid
metabolic engineering of oleaginous yeasts. Biotechnol Adv 53.
https://​doi.​org/​10.​1016/j.​biote​chadv.​2021.​107722
Chaturvedi S, Bhattacharya A, Nain L, Prasanna R, Khare SK (2019)
Valorization of agro-starchy wastes as substrates for oleaginous
microbes. Biomass Bioenerg 127:105294. https://​doi.​org/​10.​
1016/j.​biomb​ioe.​2019.​105294
Chen C, Kao A, Tsai Z, Chow T, Chang H, Zhao X, Chen P, Su H,
Chang J (2016a) Expression of type 2 diacylglycerol acyltrans-
ferse gene DGTT1 from Chlamydomonas reinhardtii enhances
lipid production in Scenedesmus obliquus. Biotechnol J 11:336–
344. https://​doi.​org/​10.​1002/​biot.​20150​0272
Chen W, Zhou PP, Zhu YM, Xie C, Ma L, Wang XP, Bao ZD, Yu
LJ (2016b) Improvement in the docosahexaenoic acid produc-
tion of Schizochytrium sp S056 by replacement of sea salt.
Bioproc Biosyst Eng 39:315–321. https://​d oi.​o rg/​1 0.​1 007/​
s00449-​015-​1517-1
Chen J, Zhang X, Yan S, Tyagi R, Drogui P (2017) Lipid production
from fed-batch fermentation of crude glycerol directed by the
kinetic study of batch fermentations. Fuel 209:1–9. https://​doi.​
org/​10.​1016/j.​fuel.​2017.​07.​070
Chen J, Zhang X, Drogui P, Tyagi RD (2018) The pH-based fed-batch
for lipid production from Trichosporon oleaginosus with crude
glycerol. Bioresour Technol 259:237–243. https://​doi.​org/​10.​
1016/j.​biort​ech.​2018.​03.​045
13
79670
Chen L, Qian X, Zhang X, Zhou X, Zhou J, Dong W, Xin F, Zhang W,
Jiang M, Ochsenreither K (2021) Co-production of microbial
lipids with valuable chemicals. Biofuel Bioprod Bior 15:945–
954. https://​doi.​org/​10.​1002/​bbb.​2209
Chi Z, Zheng Y, Jiang A, Chen S (2011) Lipid production by cultur-
ing oleaginous yeast and algae with food waste and municipal
wastewater in an integrated process. Appl Biochem Biotechnol
165:442–453. https://​doi.​org/​10.​1007/​s12010-​011-​9263-6
Chintagunta AD, Zuccaro G, Kumar M, Kumar SPJ, Garlapati VK,
Postemsky PD, Kumar NSS, Chandel AK, Simal-Gandara J
(2021) Biodiesel production from lignocellulosic biomass using
oleaginous microbes: prospects for integrated biofuel production.
Front. Microbiol 12. https://​doi.​org/​10.​3389/​fmicb.​2021.​658284
Cho HU, Park JM (2018) Biodiesel production by various oleagi-
nous microorganisms from organic wastes. Bioresour Technol
256:502–508. https://​doi.​org/​10.​1016/j.​biort​ech.​2018.​02.​010
Cho HU, Kim YM, Park JM (2017) Enhanced microalgal biomass and
lipid production from a consortium of indigenous microalgae
and bacteria present in municipal wastewater under gradually
mixotrophic culture conditions. Bioresour Technol 228:290–297.
https://​doi.​org/​10.​1016/j.​biort​ech.​2016.​12.​094
Cho IJ, Choi KR, Lee SY (2020) Microbial production of fatty acids
and derivative chemicals. Curr Opin Biotechnol 65:129–141.
https://​doi.​org/​10.​1016/j.​copbio.​2020.​02.​006
Christophe G, Kumar V, Nouaille R, Gaudet G, Fontanille P, Pandey
A, Soccol CR, Larroche C (2012) Recent developments in micro-
bial oils production: a possible alternative to vegetable oils for
biodiesel without competition with human food? Braz Arch Biol
Technol 55:29–46. https://​doi.​org/​10.​1590/​S1516-​89132​01200​
01000​04
Cui Y, Zhao J, Wang Y, Qin S, Lu Y (2018) Characterization and
engineering of a dual-function diacylglycerol acyltransferase in
the oleaginous marine diatom Phaeodactylum tricornutum. Bio-
technol Biofuels 11. https://​doi.​org/​10.​1186/​s13068-​018-​1029-8
Daboussi F, Leduc S, Marechal A, Dubois G, Guyot V, Perez-Michaut
C, Amato A, Falciatore A, Juillerat A, Beurdeley M, Voytas DF,
Cavarec L, Duchateau P (2014) Genome engineering empowers
the diatom Phaeodactylum tricornutum for biotechnology. Nat
Commun 5:3831. https://​doi.​org/​10.​1038/​ncomm​s4831
Das S, Nath K, Chowdhury R (2021) Comparative studies on biomass
productivity and lipid content of a novel blue-green algae during
autotrophic and heterotrophic growth. Environ Sci Pollut Res
28:12107–12118. https://​doi.​org/​10.​1007/​s11356-​020-​09577-4
Das M, Patra P, Ghosh A (2020) Metabolic engineering for enhancing
microbial biosynthesis of advanced biofuels. Renew Sust Energ
Rev 119. https://​doi.​org/​10.​1016/j.​rser.​2019.​109562
Davies RJ, Holdsworth JE, Reader SL (1990) The effect of low oxy-
gen uptake rate on the fatty acid profile of the oleaginous yeast
Apiotrichum curvatum. Appl Microbiol Biotechnol 33:569–573
Demidenko A, Akberdin IR, Allemann M, Allen EE, Kalyuzhnaya MG
(2017) Fatty acid biosynthesis pathways in methylomicrobium
buryatense 5G(B1). Front Microbiol 7. https://​doi.​org/​10.​3389/​
fmicb.​2016.​02167
Donot F, Fontana A, Baccou J, Strub C, Schorr-Galindo S (2014) Sin-
gle cell oils (SCOs) from oleaginous yeasts and moulds: produc-
tion and genetics. Biomass Bioenerg 68:135–150. https://d​ oi.o​ rg/​
10.​1016/j.​biomb​ioe.​2014.​06.​016
Dourou M, Aggeli D, Papanikolaou S, Aggelis G (2018) Critical steps
in carbon metabolism affecting lipid accumulation and their reg-
ulation in oleaginous microorganisms. Appl Microbiol Biotech-
nol 102:2509–2523. https://​doi.​org/​10.​1007/​s00253-​018-​8813-z
Dulermo T, Treton B, Beopoulos A, Gnankon APK, Haddouche R,
Nicaud JM (2013) Characterization of the two intracellular
lipases of Y. lipolytica encoded by TGL3 and TGL4 genes:
new insights into the role of intracellular lipases and lipid body
13
Environmental Science and Pollution Research (2023) 30:79654–79675
organisation. B Mol Cell Biol L 1831:1486–1495. https://​doi.​
org/​10.​1016/j.​bbalip.​2013.​07.​001
Eungrasamee K, Miao R, Incharoensakdi A, Lindblad P, Jantaro S
(2019) Improved lipid production via fatty acid biosynthesis
and free fatty acid recycling in engineered Synechocystis sp.
PCC 6803. Biotechnol Biofuels 12. https://​doi.​org/​10.​1186/​
s13068-​018-​1349-8
Fei Q, Wewetzer SJ, Kurosawa K, Rha C, Sinskey AJ (2015) High-
cell-density cultivation of an engineered Rhodococcus opacus
strain for lipid production via co-fermentation of glucose and
xylose. Process Biochem 50:500–506. https://​doi.​org/​10.​1016/j.​
procb​io.​2015.​01.​008
Fei Q, O’Brien M, Nelson R, Chen X, Lowell A, Dowe N (2016)
Enhanced lipid production by Rhodosporidium toruloides using
different fed-batch feeding strategies with lignocellulosic hydro-
lysate as the sole carbon source. Biotechnol Biofuels 9:1–12.
https://​doi.​org/​10.​1186/​s13068-​016-​0542-x
Fontanille P, Kumar V, Christophe G, Nouaille R, Larroche C (2012)
Bioconversion of volatile fatty acids into lipids by the oleagi-
nous yeast Yarrowia lipolytica. Bioresour Technol 114:443–449.
https://​doi.​org/​10.​1016/j.​biort​ech.​2012.​02.​091
Fu R, Fei Q, Shang L, Brigham CJ, Chang HN (2018) Enhanced micro-
bial lipid production by Cryptococcus albidus in the high-cell-
density continuous cultivation with membrane cell recycling
and two-stage nutrient limitation. J Ind Microbiol Biotechnol
45:1045–1051. https://​doi.​org/​10.​1007/​s10295-​018-​2081-x
Galafassi S, Cucchetti D, Pizza F, Franzosi G, Bianchi D, Compagno
C (2012) Lipid production for second generation biodiesel by
the oleaginous yeast Rhodotorula graminis. Bioresour Technol
111:398–403. https://​doi.​org/​10.​1016/j.​biort​ech.​2012.​02.​004
Ganuza E, Izquierdo MS (2007) Lipid accumulation in Schizochytrium
G13/2S produced in continuous culture. Appl Microbiol Bio-
technol 76:985–990. https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00253-0​ 07-1​ 019-4
Gao Z, Ma Y, Wang Q, Zhang M, Wang J, Liu Y (2016) Effect of crude
glycerol impurities on lipid preparation by Rhodosporidium toru-
loides yeast 32489. Bioresour Technol 218:373–379. https://​doi.​
org/​10.​1016/j.​biort​ech.​2016.​06.​088
Gao Z, Ma YQ, Ma XY, Wang QH, Liu Y (2019) A novel variable pH
control strategy for enhancing lipid production from food waste:
biodiesel versus docosahexaenoic acid. Energy Convers Man-
age 189:60–66. https://​doi.​org/​10.​1016/j.​encon​man.​2019.​03.​078
Gao Z, Ma Y, Liu Y, Wang Q (2022) Waste cooking oil used as car-
bon source for microbial lipid production: promoter or inhibitor.
Environ Res 203:111881. https://​doi.​org/​10.​1016/j.​envres.​2021.​
111881
Garay LA, Sitepu IR, Cajka T, Chandra I, Shi S, Lin T, German JB,
Fiehn O, Boundy-Mills KL (2016) Eighteen new oleaginous
yeast species. J Ind Microbial Biot 43:887–900. https://​doi.​org/​
10.​1007/​s10295-​016-​1765-3
Garlapati VK, Chandel AK, Kumar SPJ, Sharma S, Sevda S, Ingle
AP, Pant D (2020) Circular economy aspects of lignin: towards
a lignocellulose biorefinery. Renew Sust Energ Rev 130. https://​
doi.​org/​10.​1016/j.​rser.​2020.​109977
Ge C, Chen H, Mei T, Tang X, Chang L, Gu Z, Zhang H, Chen W,
Chen YQ (2018) Application of a ω-3 desaturase with an ara-
chidonic acid preference to eicosapentaenoic acid production in
Mortierella alpina. Front Bioeng Biotechnol 5:89. https://d​ oi.o​ rg/​
10.​3389/​fbioe.​2017.​00089
Gong Z, Shen H, Zhou W, Wang Y, Yang X, Zhao Z (2015) Efficient
conversion of acetate into lipids by the oleaginous yeast Crypto-
coccus curvatus. Biotechnol Biofuels 8. https://​doi.​org/​10.​1186/​
s13068-​015-​0371-3
Gong Z, Wang X, Yuan W, Wang Y, Zhou W, Wang G, Liu Y (2020)
Fed-batch enzymatic hydrolysis of alkaline organosolv-pre-
treated corn stover facilitating high concentrations and yields
Environmental Science and Pollution Research (2023) 30:79654–79675
of fermentable sugars for microbial lipid production. Biotech-
nol Biofuels 13. https://​doi.​org/​10.​1186/​s13068-​019-​1639-9
Gorte O, Kugel M, Ochsenreither K (2020) Optimization of carbon
source efficiency for lipid production with the oleaginous yeast
Saitozyma podzolica DSM 27192 applying automated continu-
ous feeding. Biotechnol Biofuels 13:181. https://​doi.​org/​10.​
1186/​s13068-​020-​01824-7
Goswami L, Namboodiri MMT, Kumar RV, Pakshirajan K, Pugaz-
henthi G (2017) Biodiesel production potential of oleaginous
Rhodococcus opacusgrown on biomass gasification wastewa-
ter. Renewable Energy 105:400–406. https://​doi.​org/​10.​1016/j.​
renene.​2016.​12.​044
Gujjala LKS, Kumar SPJ, Talukdar B, Dash A, Kumar S, Sherpa
KC, Banerjee R (2019) Biodiesel from oleaginous microbes:
opportunities and challenges. Biofuels-UK 10:45–59. https://​
doi.​org/​10.​1080/​17597​269.​2017.​14025​87
Han X, Zhao Z, Wen Y, Chen Z (2020) Enhancement of docosahex-
aenoic acid production by overexpression of ATP-citrate lyase
and acetyl-CoA carboxylase in Schizochytrium sp. Biotechnol
Biofuels 13. https://​doi.​org/​10.​1186/​s13068-​020-​01767-z
Huang C, Chen X, Xiong L, Ma L, Chen Y (2013) Single cell oil pro-
duction from low-cost substrates: the possibility and potential
of its industrialization. Biotechnol Adv 31:129–139. https://​
doi.​org/​10.​1016/j.​biote​chadv.​2012.​08.​010
Huang C, Guo HJ, Xiong L, Wang B, Shi SL, Chen XF, Lin XQ,
Wang C, Luo J, Chen XD (2016a) Using wastewater after lipid
fermentation as substrate for bacterial cellulose production by
Gluconacetobacter xylinus. Carbohydr Polym 136:198–202.
https://​doi.​org/​10.​1016/j.​carbp​ol.​2015.​09.​043
Huang XF, Liu JN, Lu LJ, Peng KM, Yang GX, Liu J (2016b) Cul-
ture strategies for lipid production using acetic acid as sole car-
bon source by Rhodosporidium toruloides. Bioresour Technol
206:141–149. https://​doi.​org/​10.​1016/j.​biort​ech.​2016.​01.​073
Huang C, Luo M-T, Chen X-F, Qi G-X, Xiong L, Lin X-Q, Wang C,
Li H-L, Chen X-D (2017) Combined “de novo” and “ex novo”
lipid fermentation in a mix-medium of corncob acid hydro-
lysate and soybean oil by Trichosporon dermatis. Biotechnol
Biofuels 10. https://​doi.​org/​10.​1186/​s13068-​017-​0835-8
Humaidah N, Nakai S, Nishijima W, Gotoh T, Furuta M (2020)
Application of Aurantiochytrium sp. L3W for food-process-
ing wastewater treatment in combination with polyunsaturated
fatty acids production for fish aquaculture. Sci Total Environ
743. https://​doi.​org/​10.​1016/j.​scito​tenv.​2020.​140735
Iwai M, Ikeda K, Shimojima M, Ohta H (2014) Enhancement of
extraplastidic oil synthesis in C hlamydomonas reinhardtii
using a type-2 diacylglycerol acyltransferase with a phosphorus
starvation–inducible promoter. Plant Biotechnol J 12:808–819.
https://​doi.​org/​10.​1111/​pbi.​12210
Jakhwal P, Biswas JK, Tiwari A, Kwon EE, Bhatnagar A (2022)
Genetic and non-genetic tailoring of microalgae for the
enhanced production of eicosapentaenoic acid (EPA) and doco-
sahexaenoic acid (DHA) - a review. Bioresour Technol 344.
https://​doi.​org/​10.​1016/j.​biort​ech.​2021.​126250
Jeevan Kumar S, Avanthi A, Chintagunta AD, Gupta A, Banerjee R
(2020) Oleaginous lipid: a drive to synthesize and utilize as
biodiesel, Practices and Perspectives in Sustainable Bioenergy.
Springer, pp. 105–129
Jeon S, Koh HG, Cho JM, Kang NK, Chang YK (2021) Enhancement
of lipid production in Nannochloropsis salina by overexpres-
sion of endogenous NADP-dependent malic enzyme. Algal Res
54. https://​doi.​org/​10.​1016/j.​algal.​2021.​102218
Jin M, Slininger PJ, Dien BS, Waghmode S, Moser BR, Orjuela A,
Sousa Lda C, Balan V (2015) Microbial lipid-based lignocellu-
losic biorefinery: feasibility and challenges. Trends Biotechnol
33:43–54. https://​doi.​org/​10.​1016/j.​tibte​ch.​2014.​11.​005
79671
Kamineni A, Shaw J (2020) Engineering triacylglycerol production
from sugars in oleaginous yeasts. Curr Opin Biotechnol 62:239–
247. https://​doi.​org/​10.​1016/j.​copbio.​2019.​12.​022
Kang NK, Baek K, Koh HG, Atkinson CA, Ort DR, Jin YS (2022)
Microalgal metabolic engineering strategies for the production
of fuels and chemicals. Bioresour Technol 345. https://​doi.​org/​
10.​1016/j.​biort​ech.​2021.​126529
Karamerou EE, Theodoropoulos C, Webb C (2016) A biorefinery
approach to microbial oil production from glycerol by Rhodoto-
rula glutinis. Biomass Bioenerg 89:113–122. https://​doi.​org/​10.​
1016/j.​biomb​ioe.​2016.​01.​007
Karamerou EE, Webb C (2019) Cultivation modes for microbial oil
production using oleaginous yeasts – a review. Biochem Eng J
151. https://​doi.​org/​10.​1016/j.​bej.​2019.​107322
Karmakar B, Mukherjee S, Sengupta SL, Halder G (2021) Recircu-
lating used cooking oil and Nagkesar seed shells in dual-stage
catalytic biodiesel synthesis with C-1-C-3 alcohols. Envi-
ron Sci Pollut Res 28:58154–58169. https://​doi.​org/​10.​1007/​
s11356-​021-​14309-3
Karnaouri A, Chalima A, Kalogiannis KG, Varamogianni-Mamatsi
D, Lappas A, Topakas E (2020) Utilization of lignocellulosic
biomass towards the production of omega-3 fatty acids by the
heterotrophic marine microalga Crypthecodinium cohnii. Biore-
sour Technol 303:122899. https://​doi.​org/​10.​1016/j.​biort​ech.​
2020.​122899
Karnaouri A, Asimakopoulou G, Kalogiannis KG, Lappas AA, Topa-
kas E (2021) Efficient production of nutraceuticals and lactic acid
from lignocellulosic biomass by combining organosolv fractiona-
tion with enzymatic/ fermentative routes. Bioresour. Technol.
341. https://​doi.​org/​10.​1016/j.​biort​ech.​2021.​125846
Khot M, Raut G, Ghosh D, Alarcon-Vivero M, Contreras D, Ravikumar
A (2020) Lipid recovery from oleaginous yeasts: perspectives
and challenges for industrial applications. Fuel 259. https://​doi.​
org/​10.​1016/j.​fuel.​2019.​116292
Kim HM, Chae TU, Choi SY, Kim WJ, Lee SY (2019) Engineering
of an oleaginous bacterium for the production of fatty acids and
fuels. Nat Chem Biol 15:721–729. https://​doi.​org/​10.​1038/​nbt.​
3763
Kong F, Liang Y, Légeret B, Beyly-Adriano A, Blangy S, Haslam
RP, Napier JA, Beisson F, Peltier G, Li-Beisson Y (2017) Chla-
mydomonas carries out fatty acid β-oxidation in ancestral peroxi-
somes using a bona fide acyl-CoA oxidase. Plant J 90:358–371.
https://​doi.​org/​10.​1111/​tpj.​13498
Konzock O, Matsushita Y, Zaghen S, Sako A, Norbeck J (2022) Alter-
ing the fatty acid profile of Yarrowia lipolytica to mimic cocoa
butter by genetic engineering of desaturases. Microb Cell Fact
21:25. https://​doi.​org/​10.​1186/​s12934-​022-​01748-x
Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes:
advances in metabolism research. Trends Biotechnol 29:53–61.
https://​doi.​org/​10.​1016/j.​tibte​ch.​2010.​11.​002
Kumar LR, Yellapu SK, Tyagi RD, Drogui P (2021) Microbial lipid
and biodiesel production from municipal sludge fortified with
crude glycerol medium using pH-based fed-batch strategy. J
Environ Chem Eng 9. https://d​ oi.o​ rg/1​ 0.1​ 016/j.j​ ece.2​ 021.1​ 05032
Kumar N, Banerjee C, Chang JS, Shukla P (2022) Valorization of
wastewater through microalgae as a prospect for generation of
biofuel and high-value products. J. Clean. Prod.: 132114. https://​
doi.​org/​10.​1016/j.​jclep​ro.​2022.​132114
Lazar Z, Liu N, Stephanopoulos G (2018) Holistic approaches in lipid
production by Yarrowia lipolytica. Trends Biotechnol 36:1157–
1170. https://​doi.​org/​10.​1016/j.​tibte​ch.​2018.​06.​007
Ledesma-Amaro R, Dulermo T, Nicaud JM (2015) Engineering Yar-
rowia lipolytica to produce biodiesel from raw starch. Biotechnol
Biofuels 8:148. https://​doi.​org/​10.​1186/​s13068-​015-​0335-7
Leong W, Lim J, Lam M, Uemura Y, Ho Y (2018) Third generation
biofuels: a nutritional perspective in enhancing microbial lipid
13
79672
production. Renew Sust Energ Rev 91:950–961. https://​doi.​org/​
10.​1016/j.​rser.​2018.​04.​066
Levering J, Broddrick J, Zengler K (2015) Engineering of oleaginous
organisms for lipid production. Curr Opin Biotechnol 36:32–39.
https://​doi.​org/​10.​1016/j.​copbio.​2015.​08.​001
Li Y, Zhao Z, Bai F (2007) High-density cultivation of oleaginous
yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzym
Microb Technol 41:312–317. https://​doi.​org/​10.​1016/j.​enzmi​
ctec.​2007.​02.​008
Li Y, Han D, Hu G, Dauvillee D, Sommerfeld M, Ball S, Hua Q (2010)
Chlamydomonas starchless mutant defective in ADP-glucose
pyrophosphorylase hyper-accumulates triacylglycerol. Metab
Eng 12:387–391. https://​doi.​org/​10.​1016/j.​ymben.​2010.​02.​002
Li D, Cen S, Liu Y, Balamurugan S, Zheng X, Alimujiang A, Yang W,
Liu J, Li H (2016) A type 2 diacylglycerol acyltransferase accel-
erates the triacylglycerol biosynthesis in heterokont oleaginous
microalga Nannochloropsis oceanica. J Biotechnol 229:65–71.
https://​doi.​org/​10.​1016/j.​jbiot​ec.​2016.​05.​005
Li D, Xie W, Hao T, Cai J, Zhou T, Balamurugan S, Yang W, Liu J,
Li H (2018) Constitutive and chloroplast targeted expression of
acetyl-CoA carboxylase in oleaginous microalgae elevates fatty
acid biosynthesis. Mar Biotechnol 20:566–572. https://​doi.​org/​
10.​1007/​s10126-​018-​9841-5
Liang M, Jiang J (2013) Advancing oleaginous microorganisms to
produce lipid via metabolic engineering technology. Prog Lipid
Res 52:395–408. https://​doi.​org/​10.​1016/j.​plipr​es.​2013.​05.​002
Lin J, Shen H, Tan H, Zhao X, Wu S, Hu C, Zhao ZK (2011) Lipid pro-
duction by Lipomyces starkeyi cells in glucose solution without
auxiliary nutrients. J Biotechnol 152:184–188. https://​doi.​org/​
10.​1016/j.​jbiot​ec.​2011.​02.​010
Liu J, Yin J, Ge Y, Han H, Liu M, Gao F (2021) Improved lipid pro-
ductivity of Scenedesmus obliquus with high nutrient removal
efficiency by mixotrophic cultivation in actual municipal waste-
water. Chemosphere 285:131475. https://​doi.​org/​10.​1016/j.​
chemo​sphere.​2021.​131475
Lopes HS, Bonturi N, Kerkhoven EJ, Miranda EA, Lahtvee PJ (2020)
C/N ratio and carbon source-dependent lipid production profiling
in Rhodotorula toruloides. Appl Microbiol Biotechnol 104:2639–
2649. https://​doi.​org/​10.​1007/​s00253-​020-​10386-5
Ma Y, Gao Z, Wang Q, Liu Y (2018) Biodiesels from microbial oils:
opportunity and challenges. Bioresour Technol 263:631–641.
https://​doi.​org/​10.​1016/j.​biort​ech.​2018.​05.​028
Ma X, Gao Z, Gao M, Wu C, Wang Q (2019) Microbial lipid produc-
tion from food waste saccharified liquid under two-stage process.
Bioresour Technol 289:121626. https://​doi.​org/​10.​1016/j.​biort​
ech.​2019.​121626
Ma X, Zhang M, Gao Z, Gao M, Wu C, Wang Q (2021) Microbial lipid
production from banana straw hydrolysate and ethanol stillage.
Environ Sci Pollut Res 28:29357–29368. https://d​ oi.o​ rg/1​ 0.1​ 007/​
s11356-​021-​12644-z
Ma X, Gao M, Liu S, Li Y, Sun X, Wang Q (2022) An innovative
approach for reducing the water and alkali consumption in the
lactic acid fermentation via the reuse of pretreated liquid. Biore-
sour Technol 352:127108. https://​doi.​org/​10.​1016/j.​biort​ech.​
2022.​127108
Madzak C (2018) Engineering Yarrowia lipolytica for use in biotechno-
logical applications: a review of major achievements and recent
innovations. Mol Biotechnol 60:621–635. https://​doi.​org/​10.​
1007/​s12033-​018-​0093-4
Manowattana A, Techapun C, Watanabe M, Chaiyaso T (2018) Bio-
conversion of biodiesel-derived crude glycerol into lipids and
carotenoids by an oleaginous red yeast Sporidiobolus pararoseus
KM281507 in an airlift bioreactor. J Biosci Bioeng 125:59–66.
https://​doi.​org/​10.​1016/j.​jbiosc.​2017.​07.​014
McNeil BA, Stuart DT (2018) Lipomyces starkeyi: an emerging cell
factory for production of lipids, oleochemicals and biotechnology
13
Environmental Science and Pollution Research (2023) 30:79654–79675
applications. World J Microb Biot 34. https://​doi.​org/​10.​1007/​
s11274-​018-​2532-6
Mehmood MA, Shahid A, Malik S, Wang N, Javed MR, Haider MN,
Verma P, Ashraf MUF, Habib N, Syafiuddin A, Boopathy R
(2021) Advances in developing metabolically engineered micro-
bial platforms to produce fourth-generation biofuels and high-
value biochemicals. Bioresour Technol 337. https://​doi.​org/​10.​
1016/j.​biort​ech.​2021.​125510
Mendoza-Lopez MR, Velez-Martinez D, Argumedo-Delira R, Alarcon
A, Garcia-Barradas O, Sanchez-Viveros G, Ferrera-Cerrato R
(2017) Lipid extraction from the biomass of Trichoderma kon-
ingiopsis MX1 produced in a non-stirring culture for potential
biodiesel production. Environ Sci Pollut Res 24:25627–25633.
https://​doi.​org/​10.​1007/​s11356-​016-​6595-3
Mitra M, Shah F, Bharadwaj SVV, Patidar SK, Mishra S (2016) Cul-
tivation of nannochloropsis oceanica biomass rich in eicosapen-
taenoic acid utilizing wastewater as nutrient resource. Bioresour
Technol 218:1178–1186. https://d​ oi.o​ rg/1​ 0.1​ 016/j.b​ iorte​ ch.2​ 016.​
07.​083
Palazzolo MA, Garcia-Perez M (2022) Microbial lipid biosynthesis
from lignocellulosic biomass pyrolysis products. Biotechnol Adv
54:107791. https://​doi.​org/​10.​1016/j.​biote​chadv.​2021.​107791
Papanikolaou S, Aggelis G (2002) Lipid production by Yarrowia lipol-
ytica growing on industrial glycerol in a single-stage continuous
culture. Bioresour Technol 82:43–49. https://​doi.​org/​10.​1016/​
S0960-​8524(01)​00149-3
Papanikolaou S, Aggelis G (2010) Yarrowia lipolytica: a model micro-
organism used for the production of tailor-made lipids. Eur J
Lipid Sci Technol 112:639–654. https://​doi.​org/​10.​1002/​ejlt.​
20090​0197
Papanikolaou S, Aggelis G (2011) Lipids of oleaginous yeasts. Part II:
technology and potential applications. Eur J Lipid Sci Technol
113:1052–1073. https://​doi.​org/​10.​1002/​ejlt.​20110​0015
Park S, Nguyen THT, Jin E (2019) Improving lipid production by strain
development in microalgae: strategies, challenges and perspec-
tives. Bioresour Technol 292:121953. https://​doi.​org/​10.​1016/j.​
biort​ech.​2019.​121953
Patel A, Karageorgou D, Rova E, Katapodis P, Rova U, Christakopou-
los P, Matsakas L (2020) An overview of potential oleaginous
microorganisms and their role in biodiesel and omega-3 fatty
acid-based industries. Microorganisms 8. https://d​ oi.o​ rg/1​ 0.3​ 390/​
micro​organ​isms8​030434
Patel A, Matsakas L (2019) A comparative study on de novo and ex
novo lipid fermentation by oleaginous yeast using glucose and
sonicated waste cooking oil. Ultrason Sonochem 52:364–374.
https://​doi.​org/​10.​1016/j.​ultso​nch.​2018.​12.​010
Pinzi S, Leiva-Candia D, Lopez-Garcia I, Dolores Redel-Macias M,
Pilar Dorado M (2014) Latest trends in feedstocks for biodiesel
production. Biofuel Bioprod Bior 8:126–143. https://​doi.​org/​10.​
1002/​bbb.​1435
Polburee P, Limtong S (2020) Economical lipid production from crude
glycerol using Rhodosporidiobolus fluvialis DMKU-RK253 in
a two-stage cultivation under non-sterile conditions. Biomass
Bioenerg 138:105597. https://​doi.​org/​10.​1016/j.​biomb​ioe.​2020.​
105597
Polburee P, Yongmanitchai W, Honda K, Ohashi T, Yoshida T, Fujiy-
ama K, Limtong S (2016) Lipid production from biodiesel-
derived crude glycerol by Rhodosporidium fluviale DMKU-
RK253 using temperature shift with high cell density. Biochem
Eng J 112:208–218. https://​doi.​org/​10.​1016/j.​bej.​2016.​04.​024
Polburee P, Ohashi T, Tsai Y-Y, Sumyai T, Lertwattanasakul N, Lim-
tong S, Fujiyama K (2018) Molecular cloning and overexpression
of DGA1, an acyl-CoA-dependent diacylglycerol acyltransferase,
in the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-
RK253. Microbial-SGM 164:1–10. https://​doi.​org/​10.​1099/​
mic.0.​000584
Environmental Science and Pollution Research (2023) 30:79654–79675
Polishchuk A, Valev D, Tarvainen M, Mishra S, Kinnunen V, Antal
T, Yang B, Rintala J, Tyystjarvi E (2015) Cultivation of nanno-
chloropsis for eicosapentaenoic acid production in wastewaters of
pulp and paper industry. Bioresour Technol 193:469–476. https://​
doi.​org/​10.​1016/j.​biort​ech.​2015.​06.​135
Poontawee R, Limtong S (2020) Feeding strategies of two-stage fed-
batch cultivation processes for microbial lipid production from
sugarcane top hydrolysate and crude glycerol by the oleaginous
red yeast Rhodosporidiobolus fluvialis. Microorganisms 8.
https://​doi.​org/​10.​3390/​micro​organ​isms8​020151
Probst KV, Schulte LR, Durrett TP, Rezac ME, Vadlani PV (2016)
Oleaginous yeast: a value-added platform for renewable oils. Crit
Rev Biotechnol 36:942–955. https://​doi.​org/​10.​3109/​07388​551.​
2015.​10648​55
Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson
N, Kumaran Ajikumar P, Stephanopoulos G (2015) Engineering
lipid overproduction in the oleaginous yeast Yarrowia lipolytica.
Metab Eng 29:56–65. https://​doi.​org/​10.​1016/j.​ymben.​2015.​02.​
005
Qin L, Liu L, Zeng AP, Wei D (2017) From low-cost substrates to sin-
gle cell oils synthesized by oleaginous yeasts. Bioresour Technol
245:1507–1519. https://​doi.​org/​10.​1016/j.​biort​ech.​2017.​05.​163
Quarterman JC, Slininger PJ, Hector RE, Dien BS (2018) Engineer-
ing Candida phangngensis-an oleaginous yeast from the Yar-
rowia clade-for enhanced detoxification of lignocellulose-derived
inhibitors and lipid overproduction. FEMS Yeast Res 18. https://​
doi.​org/​10.​1093/​femsyr/​foy102
Rakicka M, Lazar Z, Dulermo T, Fickers P, Nicaud JM (2015) Lipid
production by the oleaginous yeast Yarrowia lipolytica using
industrial by-products under different culture conditions. Bio-
technol Biofuels 8. https://​doi.​org/​10.​1186/​s13068-​015-​0286-z
Raut G, Jagtap S, Kumar VR, RaviKumar A (2022) Enhancing lipid
content of oleaginous Yarrowia lipolytica biomass grown on
waste cooking oil and its conversion to biodiesel by statistical
optimization. Biomass Convers Biorefin: 1–18. https://​doi.​org/​
10.​1007/​s13399-​022-​02610-1
Rodionova MV, Bozieva AM, Zharmukhamedov SK, Leong YK, Lan
JC-W, Veziroglu A, Veziroglu TN, Tomo T, Chang J-S, Allakh-
verdiev SI (2022) A comprehensive review on lignocellulosic
biomass biorefinery for sustainable biofuel production. Int J
Hydrogen Energy 47:1481–1498. https://​doi.​org/​10.​1016/j.​ijhyd​
ene.​2021.​10.​122
Ruan Z, Zanotti M, Zhong Y, Liao W, Ducey C, Liu Y (2013) Co-
hydrolysis of lignocellulosic biomass for microbial lipid accu-
mulation. Biotechnol Bioeng 110:1039–1049. https://​doi.​org/​10.​
1002/​bit.​24773
Runguphan W, Keasling JD (2014) Metabolic engineering of Saccha-
romyces cerevisiae for production of fatty acid-derived biofuels
and chemicals. Metab Eng 21:103–113. https://d​ oi.o​ rg/1​ 0.1​ 016/j.​
ymben.​2013.​07.​003
Ryu BG, Kim J, Kim K, Choi YE, Han JI, Yang JW (2013) High-cell-
density cultivation of oleaginous yeast Cryptococcus curvatus
for biodiesel production using organic waste from the brewery
industry. Bioresour Technol 135:357–364. https://​doi.​org/​10.​
1016/j.​biort​ech.​2012.​09.​054
Saha R, Mukhopadhyay M (2021) Prospect of metabolic engineering
in enhanced microbial lipid production: review. Biomass Convers
Biorefin: 1–22. https://​doi.​org/​10.​1007/​s13399-​021-​02114-4
Sakarika M, Kornaros M (2019) Chlorella vulgaris as a green biofuel
factory: comparison between biodiesel, biogas and combustible
biomass production. Bioresour Technol 273:237–243. https://d​ oi.​
org/​10.​1016/j.​biort​ech.​2018.​11.​017
Salehmin MNI, Annuar MSM, Chisti Y (2013) High cell density fed-
batch fermentations for lipase production: feeding strategies and
oxygen transfer. Bioprocess Biosyst Eng 36:1527–1543. https://​
doi.​org/​10.​1007/​s00449-​013-​0943-1
79673
Santamauro F, Whiffin FM, Scott RJ, Chuck CJ (2014) Low-cost lipid
production by an oleaginous yeast cultured in non-sterile condi-
tions using model waste resources. Biotechnol Biofuels 7. https://​
doi.​org/​10.​1186/​1754-​6834-7-​34
Santek MI, Beluhan S, Santek B (2018) Production of microbial lipids
from lignocellulosic biomass. Adv Biofuels Bioenergy: 137–164
Santos C, Lucas MS, Dias AA, Bezerra RMF, Peres JA, Sampaio A
(2014) Winery wastewater treatment by combination of Crypto-
coccus laurentii and Fenton’s reagent. Chemosphere 117:53–58.
https://​doi.​org/​10.​1016/j.​chemo​sphere.​2014.​05.​083
Shen H, Gong Z, Yang X, Jin G, Bai F, Zhao Z (2013) Kinetics of
continuous cultivation of the oleaginous yeast Rhodosporidium
toruloides. J Biotechnol 168:85–89. https://​doi.​org/​10.​1016/j.​
jbiot​ec.​2013.​08.​010
Shen H, Li Q, Yu X (2020) Lipid production by Rhodotorula gluti-
nis in continuous cultivation with a gravity sedimentation sys-
tem. Indian J Microbial 60:246–250. https://​doi.​org/​10.​1007/​
s12088-​019-​00849-3
Shields-Menard SA, Amirsadeghi M, French WT, Boopathy R (2018)
A review on microbial lipids as a potential biofuel. Bioresour
Technol 259:451–460. https://​doi.​org/​10.​1016/j.​biort​ech.​2018.​
03.​080
Shin YS, Jeong J, Nguyen THT, Kim JYH, Jin E, Sim SJ (2019) Tar-
geted knockout of phospholipase A2 to increase lipid produc-
tivity in Chlamydomonas reinhardtii for biodiesel production.
Bioresour Technol 271:368–374. https://​doi.​org/​10.​1016/j.​biort​
ech.​2018.​09.​121
Silverman AM, Qiao K, Xu P, Stephanopoulos G (2016) Functional
overexpression and characterization of lipogenesis-related
genes in the oleaginous yeast Yarrowia lipolytica. Appl Micro-
biol Biotechnol 100:3781–3798. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 007/​
s00253-​016-​7376-0
Singh S, Bharadwaj T, Verma D, Dutta K (2022) Valorization of phenol
contaminated wastewater for lipid production by Rhodosporid-
ium toruloides 9564(T). Chemosphere 308. https://​doi.​org/​10.​
1016/j.​chemo​sphere.​2022.​136269
Sitepu IR, Garay LA, Sestric R, Levin D, Block DE, German JB,
Boundy-Mills KL (2014) Oleaginous yeasts for biodiesel: cur-
rent and future trends in biology and production. Biotechnol Adv
32:1336–1360. https://d​ oi.o​ rg/1​ 0.1​ 016/j.b​ iotec​ hadv.2​ 014.0​ 8.0​ 03
Spagnuolo M, Yaguchi A, Blenner M (2019) Oleaginous yeast for
biofuel and oleochemical production. Curr Opin Biotechnol
57:73–81. https://​doi.​org/​10.​1016/j.​copbio.​2019.​02.​011
Steiger MG, Rassinger A, Mattanovich D, Sauer M (2019) Engineering
of the citrate exporter protein enables high citric acid production
in Aspergillus niger. Metab Eng 52:224–231. https://​doi.​org/​10.​
1016/j.​ymben.​2018.​12.​004
Sundaramahalingam MA, Sivashanmugam P, Rajeshbanu J, Ashokku-
mar M (2022) A review on contemporary approaches in enhanc-
ing the innate lipid content of yeast cell. Chemosphere 293.
https://​doi.​org/​10.​1016/j.​chemo​sphere.​2022.​133616
Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid
biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel
production. Metab Eng 15:1–9. https://​doi.​org/​10.​1016/j.​ymben.​
2012.​08.​007
Takaku H, Matsuzawa T, Yaoi K, Yamazaki H (2020) Lipid metabo-
lism of the oleaginous yeastLipomyces starkeyi. Appl Micro-
biol Biotechnol 104:6141–6148. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 007/​
s00253-​020-​10695-9
Tan X, Zhang Y, Zhao X, Yang L, Yangwang S, Zou Y, Lu J (2022)
Anaerobic digestates grown oleaginous microalgae for pollutants
removal and lipids production. Chemosphere 308. https://d​ oi.o​ rg/​
10.​1016/j.​chemo​sphere.​2022.​136177
Tang M, Zhou W, Liu Y, Yan J, Gong Z (2018) A two-stage process
facilitating microbial lipid production from N-acetylglucosamine
by Cryptococcus curvatus cultured under non-sterile conditions.
13
79674
Bioresour Technol 258:255–262. https://​doi.​org/​10.​1016/j.​biort​
ech.​2018.​03.​015
Tchakouteu SS, Chatzifragkou A, Kalantzi O, Koutinas AA, Aggelis G,
Papanikolaou S (2015a) Oleaginous yeast Cryptococcus curvatus
exhibits interplay between biosynthesis of intracellular sugars
and lipids. Eur J Lipid Sci Technol 117:657–672. https://​doi.​org/​
10.​1002/​ejlt.​20140​0347
Tchakouteu SS, Kalantzi O, Gardeli C, Koutinas AA, Aggelis G,
Papanikolaou S (2015b) Lipid production by yeasts growing on
biodiesel-derived crude glycerol: strain selection and impact of
substrate concentration on the fermentation efficiency. J Appl
Microbiol 118:911–927. https://​doi.​org/​10.​1111/​jam.​12736
Tchakouteu SS, Kopsahelis N, Chatzifragkou A, Kalantzi O, Stoforos
NG, Koutinas AA, Aggelis G, Papanikolaou S (2017) Rho-
dosporidium toruloides cultivated in NaCl-enriched glucose-
based media: adaptation dynamics and lipid production. Eng
Life Sci 17:237–248. https://​doi.​org/​10.​1002/​elsc.​20150​0125
Tomas-Pejo E, Morales-Palomo S, Gonzalez-Fernandez C (2021)
Microbial lipids from organic wastes: outlook and challenges.
Bioresour Technol 323:124612. https://​doi.​org/​10.​1016/j.​biort​
ech.​2020.​124612
Tossavainen M, Lahti K, Edelmann M, Eskola R, Lampi A-M, Piironen
V, Korvonen P, Ojala A, Romantschuk M (2019) Integrated utili-
zation of microalgae cultured in aquaculture wastewater: waste-
water treatment and production of valuable fatty acids and toco-
pherols. J Appl Phycol 31:1753–1763. https://d​ oi.​org/​10.​1007/​
s10811-​018-​1689-6
Tsigie YA, Wang C-Y, Kasim NS, Diem Q-D, Huynh L-H, Ho Q-P,
Truong C-T, Ju Y-H (2012) Oil production from Yarrowia lipo-
lytica Po1g using rice bran hydrolysate. J Biomed Biotechnol
2012. https://​doi.​org/​10.​1155/​2012/​378384
Udayan A, Sabapathy H, Arumugam M (2020) Stress hormones medi-
ated lipid accumulation and modulation of specific acids in oce-
anica CASA CC201. Bioresour Technol 310. https://​doi.​org/​10.​
1016/j.​biort​ech.​2020.​123437
Vyas S, Matsakas L, Rova U, Christakopoulos P, Patel A (2023)
Insights into hydrophobic waste valorization for the production
of value-added oleochemicals. Microb Biotechnol 16:177–183.
https://​doi.​org/​10.​1111/​1751-​7915.​14122
Wang J, Xu R, Wang R, Haque ME, Liu A (2016) Overexpression of
ACC gene from oleaginous yeast Lipomyces starkeyi enhanced
the lipid accumulation in Saccharomyces cerevisiae with
increased levels of glycerol 3-phosphate substrates. Biosci Bio-
technol Biochem 80:1214–1222. https://​doi.​org/​10.​1080/​09168​
451.​2015.​11368​83
Wang J, Ledesma-Amaro R, Wei Y, Ji B, Ji XJ (2020a) Metabolic engi-
neering for increased lipid accumulation in Yarrowia lipolytica
- a review. Bioresour. Technol. 313:123707. https://​doi.​org/​10.​
1016/j.​biort​ech.​2020.​123707
Wang M, Wei Y, Ji B, Nielsen J (2020b) Advances in metabolic engi-
neering of Saccharomyces cerevisiae for cocoa butter equivalent
production. Front Bioeng Biotechnol 8:594081. https://​doi.​org/​
10.​3389/​fbioe.​2020.​594081
Wang X, Dong H, Wei W, Balamurugan S, Yang W, Liu J, Li H (2018)
Dual expression of plastidial GPAT1 and LPAT1 regulates tria-
cylglycerol production and the fatty acid profile in Phaeodac-
tylum tricornutum. Biotechnol Biofuels 11. https://​doi.​org/​10.​
1186/​s13068-​018-​1317-3
Wang X, Liu S, Li R, Yang W, Liu J, Lin CSK, Balamurugan S, Li
H (2020c) TAG pathway engineering via GPAT2 concurrently
potentiates abiotic stress tolerance and oleaginicity inPhaeodac-
tylum tricornutum. Biotechnol Biofuels 13. https://​doi.​org/​10.​
1186/​s13068-​020-​01799-5
Wei H, Shi Y, Ma X, Pan Y, Hu H, Li Y, Luo M, Gerken H, Liu
J (2017) A type-I diacylglycerol acyltransferase modulates
triacylglycerol biosynthesis and fatty acid composition in the
13
Environmental Science and Pollution Research (2023) 30:79654–79675
oleaginous microalga, Nannochloropsis oceanica. Biotechnol
Biofuels 10. https://​doi.​org/​10.​1186/​s13068-​017-​0858-1
Winwood RJ (2013) Recent developments in the commercial pro-
duction of DHA and EPA rich oils from micro-algae. OCL-
Oilseeds Fats Crops Lipids 20:D604. https://​doi.​org/​10.​1051/​
ocl/​20130​30
Wu Y, Wu Z, Yang C, Yue X, Zhou A, Song X, Su B (2023) Layered
double hydroxides for phosphorus recovery from lipid-rich
waste anaerobic fermentation liquor. J Environ Manage 326.
https://​doi.​org/​10.​1016/j.​jenvm​an.​2022.​116759
Xavier M, Coradini A, Deckmann A, Franco T (2017) Lipid produc-
tion from hemicellulose hydrolysate and acetic acid by Lipo-
myces starkeyi and the ability of yeast to metabolize inhibitors.
Biochem Eng J 118:11–19. https://​doi.​org/​10.​1016/j.​bej.​2016.​
11.​007
Xu J, Zhao X, Wang W, Du W, Liu D (2012) Microbial conversion of
biodiesel byproduct glycerol to triacylglycerols by oleaginous
yeast Rhodosporidium toruloides and the individual effect of
some impurities on lipid production. Biochem Eng J 65:30–36.
https://​doi.​org/​10.​3389/​fbioe.​2020.​594081
Xu J, Zhao X, Du W, Liu D (2017) Bioconversion of glycerol into
lipids by Rhodosporidium toruloides in a two-stage process
and characterization of lipid properties. Eng Life Sci 17:303–
313. https://​doi.​org/​10.​1002/​elsc.​20160​0062
Xu M, Sun H, Yang M, Xie D, Sun X, Meng J, Wang Q, Wu C (2022)
Biodrying of biogas residue through a thermophilic bacterial
agent inoculation: insights into dewatering contribution and
microbial mechanism. Bioresour Technol 355:127256. https://​
doi.​org/​10.​1016/j.​biort​ech.​2022.​127256
Yan Q, Pfleger BF (2020) Revisiting metabolic engineering strategies
for microbial synthesis of oleochemicals. Metab Eng 58:35–46.
https://​doi.​org/​10.​1016/j.​ymben.​2019.​04.​009
Yan J, Kuang Y, Gui X, Han X, Yan Y (2019) Engineering a malic
enzyme to enhance lipid accumulation in Chlorella protothe-
coides and direct production of biodiesel from the microalgal
biomass. Biomass Bioenerg 122:298–304. https://​doi.​org/​10.​
1016/j.​biomb​ioe.​2019.​01.​046
Yen H, Liu Y, Chang J (2015) The effects of feeding criteria on the
growth of oleaginous yeast-Rhodotorula glutinis in a pilot-scale
airlift bioreactor. J Taiwan Inst Chem Eng 49:67–71. https://​doi.​
org/​10.​1016/j.​jtice.​2014.​11.​019
Zhang Y, Dube MA, McLean DD, Kates M (2003) Biodiesel produc-
tion from waste cooking oil: 1. Process design and technological
assessment. Bioresour Technol 89:1–16. https://d​ oi.o​ rg/1​ 0.1​ 016/​
S0960-​8524(03)​00040-3
Zhang Z, O’Hara IM, Mundree S, Gao B, Ball AS, Zhu N, Bai Z, Jin
B (2016) Biofuels from food processing wastes. Curr Opin Bio-
technol 38:97–105. https://d​ oi.o​ rg/1​ 0.1​ 016/j.c​ opbio.2​ 016.0​ 1.0​ 10
Zhang W, Wu J, Zhou YJ, Liu HJ, Zhang JA (2019a) Enhanced lipid
production by Rhodotorula glutinis CGMCC 2.703 using a two-
stage pH regulation strategy with acetate as the substrate. Energy
Sci Eng 7:2077–2085. https://​doi.​org/​10.​1002/​ese3.​413
Zhang X, Chen J, Wu D, Li J, Tyagi RD, Surampalli RY (2019b) Eco-
nomical lipid production from Trichosporon oleaginosus via dis-
solved oxygen adjustment and crude glycerol addition. Bioresour
Technol 273:288–296. https://​doi.​org/​10.​1016/j.​biort​ech.​2018.​
11.​033
Zhang B, Gao M, Geng J, Cheng Y, Wang X, Wu C, Wang Q, Liu
S, Cheung SM (2021a) Catalytic performance and deactivation
mechanism of a one-step sulfonated carbon-based solid-acid
catalyst in an esterification reaction. Renew Energ 164:824–832.
https://​doi.​org/​10.​1016/j.​renene.​2020.​09.​076
Zhang L, Lee JTE, Ok YS, Dai Y, Tong YW (2022) Enhancing micro-
bial lipids yield for biodiesel production by oleaginous yeast
Lipomyces starkeyi fermentation: a review. Bioresour Technol
344:126294. https://​doi.​org/​10.​1016/j.​biort​ech.​2021.​126294
Environmental Science and Pollution Research (2023) 30:79654–79675
Zhang B, Gao M, Tang W, Wang X, Wu C, Wang Q, Cheung SM, Chen
X (2023) Esterification efficiency improvement of carbon-based
solid acid catalysts induced by biomass pretreatments: intrin-
sic mechanism. Energy 263:125606. https://​doi.​org/​10.​1016/j.​
energy.​2022.​125606
Zhang L, Lim EY, Loh KC, Dai Y, Tong YW (2021b) Two-stage fer-
mentation of Lipomyces starkeyi for production of microbial
lipids and biodiesel. Microorganisms 9. https://​doi.​org/​10.​3390/​
micro​organ​isms9​081724
Zhang S, Ren Y, Ma X, Guan W, Gao M, Li Y, Wang Q, Wu C (2021c)
Effect of zero-valent iron addition on the biogas fermentation of
food waste after anaerobic preservation. J Environ Chem Eng 9.
https://​doi.​org/​10.​1016/j.​jece.​2021.​106013
Zhao X, Hu CM, Wu SG, Shen HW, Zhao ZK (2011) Lipid produc-
tion by Rhodosporidium toruloides Y4 using different substrate
feeding strategies. J Ind Microbial Biot 38:627–632. https://​doi.​
org/​10.​1007/​s10295-​010-​0808-4
79675
Zhao T, Tashiro Y, Sonomoto K (2022) Construction and meta-
bolic analysis of acetone-butanol-ethanol fermentation using
mixed acetic acid and lactic acid in wastewater. Ind Crops Prod
187:115503. https://​doi.​org/​10.​1016/j.​indcr​op.​2022.​115503
Publisher's note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds
exclusive rights to this article under a publishing agreement with the
author(s) or other rightsholder(s); author self-archiving of the accepted
manuscript version of this article is solely governed by the terms of
such publishing agreement and applicable law.
13