Trends and Novel Strategies For Enhancing Lipid Accumulation and Quality in Microalgae

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Trends and novel strategies for enhancing lipid

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DOI: 10.1016/j.rser.2015.11.001
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Renewable and Sustainable Energy Reviews 55 (2016) 1–16
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journal homepage: www.elsevier.com/locate/rser
Trends and novel strategies for enhancing lipid accumulation

and quality in microalgae
Poonam Singh 1, Sheena Kumari, Abhishek Guldhe, Rohit Misra, Ismail Rawat, Faizal Bux n
Institute for Water and Wastewater Technology, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa
art ic l e i nf o a b s t r a c t
Article history: In order to realize the potential of microalgal biodiesel there is a need for substantial impetus involving
Received 4 August 2015 interventions to radically improve lipid yields upstream. Nutrient stress and alteration to cultivation
Received in revised form conditions are commonly used lipid enhancement strategies in microalgae. The main bottleneck of
29 October 2015
applying conventional strategies is their scalability as some of these strategies incur additional cost and
Accepted 2 November 2015
energy. Novel lipid enhancement strategies have emerged to research forefront to overcome these
challenges. In this review, the latest trends in microalgal lipid enhancement strategies, possible solutions
Keywords: and future directions are critically discussed. Advanced strategies such as combined nutrient and culti-
Microalgae vation condition stress, microalgae–bacteria interactions, use of phytohormones EDTA and chemical
Biodiesel
additives, improving light conditions using LED, dyes and paints, and gene expression analysis are
Lipid
described. Molecular approaches such as metabolic and genetic engineering are emerging as the
Genetic engineering
Phytohormones potential lipid enhancing strategies. Recent advancements in gene expression studies, genetic and
Gene expression metabolic engineering have shown promising results in enhancing lipid productivity in microalgae;
Metabolic engineering however environmental risk and long term viability are still major challenges.
& 2015 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Nutrient regime and cultivation conditions to enhance lipid accumulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Effect of nutrient regimes on lipid accumulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Effect of cultivation conditions on lipid accumulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Novel approaches for enhancing lipid accumulation in microalgae by altering culture conditions (nutritional and cultivation) . . . . . . . . . . . . . . 4
3.1. Two-stage cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2. Combined nutrients and abiotic stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.3. Phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.4. Supplementation of Ethylene-diamine-tetra-acetic acid (EDTA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.5. Co-cultivation of microalgae–bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.6. Co-cultivation of microalgae–yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.7. Improving light conditions using LED, dyes and paints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.8. Chemical additives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.8.1. Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.8.2. Brefeldin A (BFA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.8.3. Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Molecular approaches for enhancing lipid accumulation and quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.1. Microalgal genome and tools for genetic transformations in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2. Genetic engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2.1. Chloroplast engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2.2. Nuclear engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
n
Corresponding author.
E-mail address: [email protected] (F. Bux).
1
Tel.: þ27 31 373 2346; fax: þ 27 31 373 2777.
http://dx.doi.org/10.1016/j.rser.2015.11.001
1364-0321/& 2015 Elsevier Ltd. All rights reserved.
2 P. Singh et al. / Renewable and Sustainable Energy Reviews 55 (2016) 1–16
4.3. Expression analysis of genes involved in lipid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4.4. Metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.4.1. Overexpression of enzymes of lipid biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.4.2. Blocking competitive pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.4.3. Altering fatty acid chain length for improved lipid quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.4.4. Lipid secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.5. Environmental risk and challenges of molecular approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5. Impacts of microalgal lipids on biodiesel properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6. Conclusion and future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1. Introduction 2.1. Effect of nutrient regimes on lipid accumulation
Microalgae have attracted significant interest from research- Nutrients such as nitrogen, iron, phosphorus, magnesium, sul-
ersas a biodiesel feedstock due to its capacity to accumulate sub- fur and silicon are very important for cellular mechanism viz,
stantial amount of lipids, high growth rate and environmental photosynthesis, cell division, respiration, intracellular transporta-
benefits. Production of value added by-products and utilization of tion, protein synthesis etc in microalgae [6,21]. Under stressed
lipid-extracted biomass (animal feed, aquaculture and biomethane conditions; microalgae tend to accumulate energy in the form of
production) have further strengthened the case for microalgae as a polysaccharides, and/or neutral lipids. This defense mechanism of
sustainable feedstock for biodiesel production. Despite many the microalgal cell has been exploited widely for the production of
advantages, commercial realization of microalgal biodiesel is still a neutral lipids, carotenoid, polysaccharides and many other meta-
challenge owing to its high production cost [1–5]. Enhancement of bolites [22–24].
microalgal lipid content could improve the economics of biodiesel Nitrogen starvation is the most widely used strategy to
production. Nutrient limitation and induction of stress by con- improve lipid accumulation. Nitrogen is provided in the form of
trolled cultivation have been the norm to improve lipid content in nitrates, urea and ammonium salts. Uptake and utilization of these
microalgae [1,6,7]. Recently, novel strategies have been explored nitrogen forms by microalgae however vary; ammonia is utilized
to overcome the challenges of conventional approachesand to more efficiently compared to the other forms of nitrogen as it can
achieve maximum possible outcomes in terms of lipid yields, be directly converted to amino acids [25–27]. Nitrogen deprivation
sustainability and cost effectiveness [8,9]. These strategies include conditions could lead to reduced cell division [28]. Reduced cell
a combination of stress factors, co-culturing with other micro- division shifts the lipid biosynthetic pathways to synthesize more
organisms, addition of phytohormones and chemical additives neutral lipids than synthesizing membrane lipids required for the
[10–13]. cell wall formation [29,30].Subsequent accumulation of NADH due
Developing microalgal strains with high lipid accumulation to the slower photosynthetic rate inhibits enzyme citrate synthase
capability using genetic and metabolic engineering tools has and prevents acetyl CoA from entering into the TCA cycle. Elevated
recentlygainedmomentum as alternative strategies for strain concentrations of acetyl CoA activate acetyl CoA carboxylase,
improvement [14,15]. The recent advancements in decoding the which converts acetyl CoA to malonyl CoA. This irreversible con-
full genome of several microalgal strains and identification of key version reaction is the rate limiting step infatty acid biosynthesis
genes involved in lipid synthesis pathways makes genetic and which leads to enhanced lipid accumulation in microalgal cells
metabolic engineering an alluring strategy to enhance lipid accu- [31]. Tao et al. [32] studied the effect of nitrogen limitaion on lipid
mulation in microalgae [16]. The major challenges for genetically yields of Chorococcum spp. and Scenedesmus destricola. Under
modified microalgal strain for high lipid accumulation are their nitrogen deficent conditions, the lipid conent was increased from
long term viability and environmental risk assessment at open 31.6% to 40.7% in Chlorococcum nivale and 48% to 54% in
cultivation systems.
The present review deals with recent advancements and novel
strategies for lipid enhancement in microalgae and their chal-
lenges.Molecular approaches for enhancing the lipid accumulation
and quality are also described in detail (Fig. 1).
2. Nutrient regime and cultivation conditions to enhance lipid

accumulation
Microalgae are able to survive in extreme environments as they

can alter their metabolism according to varied environmental
conditions. Under unfavorable conditions, microalgae have the
tendency to accumulate neutral lipids to protect cells from photo-
oxidation [7,17,18]. Lipid enhancement strategies involving
alteration of the nutrient regime and cultivation conditions are
widely applied in microalgal cultivation. Factors such as nutrient
stress, light, temperature, CO2, salinity etc. have been explored by
several researchers to enhance lipid accumulation in microalgae
[19,20]. Fig. 1. Lipid enhancement strategies for sustainable microalgal biodiesel production.
P. Singh et al. / Renewable and Sustainable Energy Reviews 55 (2016) 1–16 3
Fig. 2. Molecular strategies for enhancing lipid accumulation in microalgae.
Scenedesmus destricola. Biomass productivity of Chlorococcum biomass and lipid accumulation [11,39]. Liu et al. [40] have
nivale and Scenedesmus destricola was decreased from reported 3–7 folds increase in lipid accumulation as compare to
0.40 gL 1d 1 to 0.38 gL 1d 1 and 0.48 gL 1 d 1 to 0.38 gL 1d 1 the control in Chlorella vulgaris under higher iron concentration.
repectively under these conditions. Converti et al. [29] studied the Baky et al. [41] cultivated Scenedesmus obliquus in N-9 medium
effect of nitrogen limitation on lipid yields of Nannochloropsis with varying concentrations of FeCl3 and lipid yields were found to
oculata and C. vulgaris. Under nitrogen limited conditions, the lipid increase with an increase inFeCl3 concentration. Maximum lipid
yield was increased to 15.31% and 16.41% in Nannochloropsis ocu- yield of 28.12% was observed in culture with FeCl3 concentration of
lata and Chlorella vulgaris respectively. Similarly Gao et al. [33] 20 mgL 1.
reported increase in lipid accumulation from 23 to 46% and Similarly, Magnesium ions are also found to influence the lipid
decrease in biomass productivity from 19.0 mgL 1d 1 to accumulation in microalgae [42]. Magnesium ions promote the
12 mgL 1d 1 in Chaetoceros muelleri under nitrogen limitation. activity of acetyl-CoA, the enzyme that regulates the first com-
Altering the nitrogen source in the media may also lead to change mitting step of microalgal lipid biosynthesis and are also required
in the quantity of lipid accumulation and fatty acid composition. for chloroplast pyruvate dehydrogenase complex which provides
Lin and Lin [34] reported that a combination of urea and sodium acetyl-CoA and NADH for fatty acid synthesis [43,44]. Gorain et al.
nitrate results in highest biomass productivity in Scenedesmus [45] observed an improvement in lipid content (1.44 fold rise)
rubescens. Tao et al. [32] found that deficiency of nitrate and urea with the addition of magnesium (100 mgL 1) into the media.
results in variation of lipid content and fatty acid composition of Huang et al. [46] reported 1.25 folds increase in the cell density of
Chlorococcum ellipsoideum, Chlorococcum nivale, Chlorococcum Monoraphidium sp. FXY-10 with supplementation of 100 mM
tatrense and Scenedesmus deserticola strain (Fig. 2). magnesium into the medium. Ren et al. [43] also observed increase
Phosphorous is an important macronutrient that contributes to in lipid content and growth with increasing magnesium con-
various metabolic processes such as signaling pathways, energy centration. Therefore an increase in Mg supply to the media could
generation, and photosynthesis. Under phosphate limited culti- enhance the activity of these enzymes, leading to an increased cell
vation conditions, microalgal cells are known to accumulate neu- division under nutrient stress conditions. Table 1 represents the
tral lipids. Xin et al. [25] reported a highest lipid accumulation of conventional lipid enhancement strategies for sustainable micro-
53% under phosphate limitation (0.1 mgL 1) in Scenedesmus sp. algal biodiesel production.
however; they observed low biomass of 0.13 gL 1 under the same Nutrient regime alterations are the preferred choice for the
conditions. Microalgal cells store phosphorus as polyphosphate enhancement of lipid accumulation, because of its easy applic-
under nutrient sufficient condition which are utilized by the cell ability at both lab and large scale cultivation. However, the main
during nutrient deficient conditions [35–37]. Although the above challenge for this strategy is the trade-off between biomass and
nutrient stress strategies are successful in terms of enhancing lipid lipid yields. Nutrients like nitrogen have significant influence on
content in microalgae; low biomass production is the main lim- both the biomass generation as well as the lipid accumulation in
itation as it affects the overall lipid productivity. the cell. Increase in the nitrogen concentration in the medium
Metals such as iron are known to influence microalgal growth directly affects the biomass and inversely affects the lipid yields
under nutrient stress conditions. It is one of the most important- [17]. Nutrients such as nitrogen iron and phosphorous has been
metals in photosystem (I) and photosystem (II), and play roles in widely studied for microalgal lipid enhancement, however other
nitrogen assimilation, respiration and DNA synthesis [38]. Lipid nutrients also need to be investigated. The further in depth studies
accumulation in microalgae occurs mainlyduring the late log on actual physiological role of these nutrient stresses on the
phase. Iron supplementation reduces the time period for the onset organism for better lipid accumulation are required. Other chal-
of stationary phase of microalgal growth thereby increasing both lenge includes identification of suitable and economical sources
Table 1
Conventional lipid enhancement strategies for sustainable microalgal biodiesel production.
Stress Microalgal strain Stress levels Lipid content (%) Biomass Lipid productivity (mgL 1d 1) Reference
Nutrient stress
N Chlorella vulgaris 375 mgL 1 16.51 - 20.30 [29]
Nannochloropsis Oculata 75 mgL 1 15.51 - 16.41 [29]
Chaetoceros muelleri Nitrogen limited 46.32 12 mgL 1d 1 4 mgL 1d 1 [33]
Chlorococcum ellipsoideum 2.9 mML 1 40.5 236.9 [57]
Scenedesmus deserticola 5.8 mML 1 54.4 0.38 gL 1d 1 216.6 [57]
P Scenedesmus Sp. 0.1 mgL 1 53 0.13 gL 1 - [25]
Chlorella sp 32 mM 23.6 1.8 gL 1 15.67 [36]
Chlorella vulgaris P deficient 37.73 38.25 mgL 1d 1 19.50 [58]
Fe Scenedesmus obliquus 20 mgL 1 28.12 1.250 mgL 1d 1 95.25 [41]
Chlorella vulgaris 1.2 10 5 mol L 1 56.6 - - [40]
Mg Monoraphidium sp. FXY-10 100 mM 58 28 mgL 1d 1 16.00 [46]
Cultivation conditions
Light Nannochloropsis sp. 100 mmol m 2 s 1 31.3 [59]
Temperature Nannochloropsis oculata 20–25 °C 7.42-14.9 [29]
CO2 Scenedesmus obliquus 12% CO2 33.14 [41]
Nannochloropsis sp. 15 % CO2 1.43 g L 1 [52]
Salinity Dunaliella salina 1.0 M 67 [55]
for the nutrients such as nitrogen, phosphorous, magnesium, while an increase in temperature from 25 to 30 °C decreased the
iron etc. lipid yield from 14.71% to 5.90% for Chlorella vulgaris.
Similarly an increase in NaCl (salt stress) in the medium can
lead to an increase in lipid content in microalgae [55,48]. An
2.2. Effect of cultivation conditions on lipid accumulation
increased NaCl concentration from 0.5 to 1.0 M improved lipid
content upto 67% in D. Salina [55]. High salinity in the culture
Cultivation conditions such as light, temperature and salinity
medium creates oxidative stress to the microalgal cells which may
have known to influence microalgal growth [47–49]. Light is
induces increase in the lipid content. Increase in salt concentration
important for photosynthesis in autotrophic microalgae and var-
can also have qualitative effect on microalgal lipids. Salinity stress
iation in light intensity and photoperiod could alter the lipid
also could facilitate in maintenances of saturated fatty acid in
biosynthesis. Wahidin et al. [50] studied the effect of light inten-
higher amounts in microalgal species. Botrycococcusbraunii grown
sity and photoperiod on lipid accumulation in Nannochloropsis sp under high salinity concentrations of 34 mM and 85 mM, showed
(marine microalga) and noticed an increase in lipid accumulation 1.7–2.25 fold increase in the relative proportion of palmitic acid
of up to 31.3% after 8 day cultivation under 100 mmol m 2 s 1 light and 2 fold increases in oleic acid [48]. Considering the impact of
intensity and photoperiod of 18 h light: 6 h dark cycle. A gradual salinity on quality and quantity of lipids, this approach can may be
decrease in cell density and growth rate was also noticed when the use to enhance lipid accumulation in microalgae. This strategy
photoperiod cycles were extended to 24 h. It was reported that however, may only be feasible with halo-tolerant strains.
during low light intensities, microalgae tend to produce more Though the cultivation conditions are easy to maintain in the
polar lipids due to an increase in chloroplast membrane synthesis. lab, maintaining them at large scale is challenging and cost
A stepwise increase in light intensity on the other hand, tends to intensive. Controlling temperature and light is unfeasible in the
accumulate more neutral lipids without affecting the biomass open cultivation system and is cost intensive in the closed culti-
yield [51]. Although the light intensity has shown a significant role vation system [29,56]. Thesupply of CO2 to both, closed or open
in microalgal biomass and lipid accumulation, the approach is cultivation systems is unfeasible because of technical and eco-
expensive and energy intensive to scale up. nomical challenges. The choice of the cultivation system is also a
Supply of CO2 is crucial in autotrophic cultivation of microalgae. long discussed topic. Table 1 depicts effect of conventional
Microalgae fix CO2 via photosynthesis to form various organic approaches on biomass, lipid content and lipid productivity of
metabolites. It was observed that with the increased concentration microalgae. Each system has its own pros and cons, however, the
of CO2 supply, biomass and lipid productivities increase [28]. Jiang microalgal strain and economics of the process makes the choice
et al. [52] observed an increase in biomass productivity (0.39– easier. Further in depth investigation is required on the above
1.43 gL 1) and growth rate (0.33–0.52 d 1) of Nannochloropsis sp. discussed parameters, in order to decide on the economical, easy
after CO2 (15%) supplementation. and scalable strategy for the enhancement of lipid accumulation in
Microalgae can tolerate a wide range of temperatures microalgae (Table 2).
depending upon the strain. Most of the algal strains grow well in
the temperature range of 25–30 °C [53]. It has been well docu- 3. Novel approaches for enhancing lipid accumulation in
mented that an increase in temperature increases the saturated microalgae by altering culture conditions (nutritional and
fatty acids composition in microalgae while decrease in tempera- cultivation)
ture increases the unsaturated fatty acid composition of total
cellular lipids [29,54]. The temperature effect on microalgal spe- To improve the viability of producing lipids from microalgae, a
cies can however be purely strain specific. The effect of tempera- more efficient approach is required, focusing on reducing the
ture on lipid yields of Nannochloropsis oculata and Chlorella vul- overall production cost and improved scalability. Some of the
garis was studied by Converti et al. [29] and have reported that an novel approaches include combined nutrient and abiotic stress,
increase in temperature from 20 to 25 °C have resulted in two-fold use of phytohormones and EDTA, chemical additives and bacteria–
increase in lipid yield (7.90–14.92%) for Nannochloropsis oculata, microalgae co-cultivation etc.
Table 2
Comparison of various approaches their advantage and challenges.
Strategies Advantages Challenges
Conventional approaches
Nutrient stress
N High lipid accumulation Low biomass productivity
P High amount of saturated fatty acids Low lipid productivity
Metals High biomass Single stress factor is not sufficient to improve lipid productivity
High lipid productivity
High chlorophyll content
Light High biomass production due to efficient photosynthesis Not easy to control at open cultivation systems
High operational cost
Novel approaches
Two stage cultivation High biomass production at first stage Large scale trails are required
High lipid accumulation in second stage
Combined nutrient and abiotic High biomass and lipid productivity Large scale trials are required
Suitable fatty acid profile Need to find cheap nutrient sources
Easily scalability
Phytohormones High growth rate High cost of phytohormones
High biomass
EDTA supplementation Enhance metal uptake High concentration of EDTA may cause growth inhibition
High biomass production
Easily applied at large scale
Co-cultivation of Microalgae-bacteria High lipid productivity Bacterial population may affect the fatty acid composition
High growth Need further research to understand mechanism
Chemical additives
Azide High lipid accumulation without compromising growth Long term use is toxic to microalgal cell
High lipid productivity Variation in response from species to species
Brefeldin A High lipid accumulation with normal growth Toxic to cells after some time
High biomass production Need further research and optimization
Use of LED, dye and paints High lipid yield Cost intensive
High biomass Needs large scale trials
Need further research to understand effect on lipid accumulation
3.1. Two-stage cultivation 3.2. Combined nutrients and abiotic stress
To overcome the challenges of nutrient deprivation studies, Recent reports have presented the potential of combining
alternate two-stage cultivation was suggested by many research- nutrients and abiotic stress factors to improve the lipid pro-
ers to improve the lipid yield without affecting the biomass [60– ductivity in microalgae. To obtain maximum yields, it is imperative
62]. In two-stage cultivation, microalgae are initially grown under to have the knowledge of the synergistic effects of these factors as
nutrient-sufficient conditions to obtain maximum biomass and well as significance of each factor with regards to the lipid accu-
thereafter the cultivation conditions are altered to trigger the mulation [27,67]. Breuer et al. [51] investigated the effect of light,
accumulation of lipids. Mujtaba et al. [63] demonstrated the use of pH, and temperature on TAG accretion under nitrogen deficient
two-stage cultivation for improved lipid productivity in Chlorella condition and found pH and temperature to be major influencing
vulgaris. In their study, C. vulgaris was grown under nutrient rich factors for TAG accumulation. The highest TAG content (40%) was
conditions in the initial stage of cultivation and thereafter the obtained at pH 7 and 27.6 °C which was independent of light
variation. Similarly, Cao et al. [27] observed high lipid accumula-
cultivation conditions were altered to provide nitrogen stress.
tion in Chlorella minutissima as a result of combined influence of
They have noticed a lipid productivity of 71.1 mgL 1d 1 with two
iron, sodium chloride and nitrogen. Ji et al. [30] studied the effect
stage cultivation which was significantly higher than the control
of variation in temperature, light intensity and photoperiod on
(31.5 mgL 1d 1). Similarly, Ratha et al. [64] have successfully
Desmodesmus sp. and observed high biomass production at a
employed two-stage cultivation approach to improve the lipid
combination of temperature: 30 °C, light intensity:
productivity in Chlorella and Scenedesmus sp. Alvarez-Diaz et al.
98 mmol m 2 s 1 and photoperiod: 14:10 (L:D Light: Dark photo-
[65] obtained an increase (36.5–45.5%) in lipid accumulation using
period). Similarly, Pan et al. [54] have subjected four thermo-
two-stage cultivation of Ankistrodesmus falcatus. Yang et al. [66] tolerant Desmodesmus strains to nitrogen starvation under differ-
reported, supplementation of high concentration of NaCl at late ent temperature regimes. Two of the isolated strains showed lipid
log phase enhances lipid productivity during two-stage cultivation production of 50% with 75% of TAG content at 45 °C. A combina-
of Monoraphidium dybowskii LB50. Despite of several economical tion of low nitrogen and high iron concentration also reported to
benefits, the main limitation of the two stage cultivation is that increase lipid productivity of up to 74.07 mgL 1d 1in Ankis-
most of these results are strain specific and its efficiency may vary trodesmus falcatus [11]. The major advantage of combined strategy
from strain to strain. None of these studies have proved universal is that in this approach, one factor may compensate the negative
application of two-stage cultivation for all microalgal strain effect of the other. Moreover, this approach can easily be employed
(Table 2). for large scale production of microalgal biodiesel (Table 2).
3.3. Phytohormones 3.5. Co-cultivation of microalgae–bacteria
Plant growth hormones (Auxins, Cytokinins) have been iden- In the natural environment, microalgae co-exist with many
tified in microalgae and play important role in many metabolic other microorganisms including bacteria which may have influ-
pathways [68,69]. Phytohormones and there derivatives are ence microalgal growth [76,77]. The possible symbiotic relation-
reported to have a stimulating effect on microalgal growth (bio- ships between microalgae and bacteria cannot be ignored in large
mass) and metabolite production (i.e., carotenoid, lipid, carbohy- scale open cultivation systems. Therefore, selection and char-
drate and protein) [10,70]. These hormones have also been acterization of microalgal growth-promoting bacteria could offer a
reported to stimulate growth under unfavorable conditions such new strategy for improving industrial scale microalgal cultivation
as high salinity, drought and nutrient deficiency [71]. Park et al. [9] [78]. Do Nascimento et al. [79] investigated the effect of Rhizobium
reported an increase in the growth of Chlamydomonas reinhardtii sp, on the biomass, lipid and chlorophyll content of the microalgae
when auxins were supplemented to nitrogen limited medium. Ankistrodesmus sp. In their study, they found that co-culturing of
Similarly, diethyl amino ethyl hexanoate (DAH) and indole acetic Rhizobium sp. strain resulted in an increase in the chlorophyll
acid (IAA) have been reported to enhance growth (1.9 fold and (from 9 to 30 mgml 1) and biomass (699 to 1999 mgL 1) contents
2.5 fold respectively) and poly-unsaturated fatty acid content (56% of the microalgal strain. The overall lipid productivity of the strain
was increased to 112 mgL 1d 1 after optimization. Le Chevanton
and 59%, respectively) of Scenedesmus obliquus [10]. The addition
et al. [13] isolated 48 bacterial strains associated with marine
of phytohormones, 24-epibrassinolide (0.1 mM) and gibberellic
microalgae and studied their effects on growth of Dunaliella sp.
acid (100 mM) also facilitated a 3-fold increase in TAGs as well as 2-
Among these, two bacterial strains Alteromonas sp. and Muricauda
fold increase in biomass [70]. Jusoh et al. [72] observed a change in
sp. enhanced the growth of Dunaliella sp. during co-culturing.
the fatty acid composition of Chlorella vulgaris after supple-
These bacterial strains have the ability to demineralize organic
mentation of growth media with IAA (100 mM) where they
nitrogen which can be used by microalgae under nitrogen limited
observed an improvement in the quantity of palmitic and stearic
conditions. Zhao et al. [80] studied the effect of microalgae–bac-
acids and a decrease in linoleic and linolenic acids. High expres-
teria consortia on lipid production and CO2 fixation when culti-
sion of β-ketoacyl ACP synthase I (KAS I) gene, with an increase in vated using landfill leachate. The maximum biomass concentration
the levels of saturated fatty acids (C16:0 and C18:0) further con- of 1.58 gL 1 and highest lipid productivity of 24.8 mgL 1d 1 were
firmed the effect phytohormones on the metabolic pathways of C. obtained in 10% leachate spike ratio. Despite the potential benefits
vulgaris. observed thus far, there is scope for further research to determine
Assessment of input costs of applying phytohormones however, the degree of microalgae–bacteria interactions and mechanisms
is important for the economic viability of the microalgal biodiesel (Table 2).
production using this strategy. These are generally required in very
small amounts for inducing high biomass and lipid generation in 3.6. Co-cultivation of microalgae–yeast
microalgae. Phytohormones such as IAA and DAH are required in
very small quantity, 1.75–2.15 mgL 1 respectively, to achieve Microalgae–fungus symbiotic relation is very common in eco-
optimum biomass production in microalgae [10]. Park et al. [9] systems (such as Lichen). During symbiosis, microalgae are oxygen
analyzed the economic viability of phytohormones as compared, producers while yeast is CO2 supplier for photosynthesis. Both
to normal synthetic medium and acetate. The biomass production these strains are also used in bioremediation as they can converts
cost whilst using phytohormones was found to be 0.014US$/g complex substrates (industrial waste etc) into simpler forms.
(GA3) which was much more economical than without phyto- Mixed culture of microalgae and yeast (fungus) can be utilized to
hormone (0.024US$/g) and acetate (0.017US$/g). In another study produce lipids by utilizing waste as growth substrate. Cheirsilp
by Salama et al. [10] the costs of biomass production induced by et al. [76] cultivated Chlorella vulgaris and Rhodotorula glutinis
IAA was found to be 0.39 US$/g and DAH was 0.30 US$/g which using the effluent from sea food processing plant and obtained
were lower when compared with normal synthetic medium (0.78 higher biomass and lipid production as compared to the individual
US$/g). Application of phytohormones thus has great potential as culture of both strains. Yen et al. [81] studied the mutual effect of
sustainable, scalable and economical lipid enhancement strategies yeast Rhodotorula glutinis and Scenedesmus obliquus on biomass
(Table 2) and should be further investigated for a better under- and lipid accumulation of both yeast and microalgae. Co-culture
standing of the mechanisms and its application to large scale improved biomass upto 40–50% and lipid content about 60–70%
cultivations. respectively as compared to individual culture of both strains.
During co-cultivation yeast cells produces various acidic com-
pounds which serves as carbon source for microalgal growth.
3.4. Supplementation of Ethylene-diamine-tetra-acetic acid (EDTA)
Utilization of wastes, high biomass production, high and lipid
production and suitable fatty acid profile would make this co-
Ethylene-diamine-tetra-acetic acid (EDTA), a lewis acid with six
cultivation technique economical and efficient for large scale bio-
binding sites and reacts with metals and forms a stable ion diesel production.
structure, and has been extensively studied in phytoremediation
[73,74]. EDTA has been widely employed in microalgal growth 3.7. Improving light conditions using LED, dyes and paints
media for enhancing the solubility of metals, and there-
byfacilitating their uptake into the microalgal cells. Recently, Ren Light intensity and wavelength has great influence on micro-
et al. [43] reported an increase in the total lipid content (28.2%) algal growth and lipid accumulation. Improving the light condi-
and lipid productivity (29.7%) in microalgae Scenedesmus obli- tions optimum for achieving maximum growth and lipid yields
quuswith an increase in EDTA concentration (0–1 mgL 1) thereby has been investigated by several researchers. Use of light emitting
revealing its potential as a lipid enhancer.Their effectiveness can diodes (LED), dyes and paints are the recent strategies for pro-
further be improved by coupling EDTA supplementation with viding light of desired intensity and wavelength to microalgal
nutrient and metal stress[75]. Due to the comparatively small culture. The chlorophyll pigments absorb mainly in the range of
amount of additional EDTA required and inexpensive nature of the blue (450–475 nm) and red (630–675 nm) light [82]. The perfor-
chemical, this approach could be easily adopted at large scale. mance of photosystem I and II can be enhanced by providing the
light in the range of red and blue wavelengths respectively [83]. N,N,N-trimethylhomoserine). These results proved that Brefeldin
LED can be used to provide light with narrow band spectrum and induces stress in ER of microalgae that causes the change in lipid
has several advantages over the conventional fluorescent lamps composition and converts phospholipids and sterol into neutral
such as low power consumption, longer lifetime, low heat gen- lipids. This mechanism is independent of nitrogen stress induced
eration, high conversion efficiency. The life time of LED are 941% lipid accumulation. Thus, this strategy can improve lipid accu-
and intensity (Wm 2) 500% more compared to the conventional mulation without compromising biomass and lipid productivity.
fluorescent lamps [59,83]. Atta et al. [59] investigated the effect of However, economic feasibility of utilizing this chemical and its
blue LED and conventional white lights on the lipid yields of C. toxic nature that can reduce biomass production needs further
vulgaris. They observed a maximum lipid yield of 23.5% C. vulgaris investigation before application of this method on commercial
was 23.5% in 8 days cultivation time under blue LED conditions, scale can be considered (Table 2).Thus further research is required
while the lipid content was 20.9% in 10 days cultivation time to understand the effect BFA on lipid biosynthesis pathway.
under white light conditions. Photosynthetically active radiations
(PAR) account for less than 15% of the solar radiation spectrum. 3.8.3. Surfactants
Fluorophores such as organic dyes and fluorescent paints can Surfactants are widely used in household and industrial processes.
alter the photons with high energy to the low energy light These compounds denature the proteins in cell wall structure and alter
wavelengths [84,85]. Seo et al. [84] found that a mixture of rho- its permeability. Polyoxyethylenesorbitanmonooleate (Tween-80) is
damine 101 and 9, 10-diphenylanthracene dye solution improved non-ionic surfactant which is known to improve nutrient availability
the lipid productivity of C. vulgaris by 2.3 times compared to the to cell by improving permeability of cell membrane. Taoka et al. [90]
cultivation without using dye. In their study, they have also con- observed supplementation of 1% tween-80 in the medium lead to a
firmed that individually these dyes have different effect on bio- 2 fold increase in biomass and 1.15 fold increase in lipid content of
mass and lipid production. Seo et al. [85] studied the effect of marine protist Thraustochytrium aureum ATCC 34304. This approach
yellow, red, blue and green florescent paint solutions on the
can be applied to microalgae for enhanced lipid accumulation. This
growth and lipid accumulation of Chlorella sp. In their study they
approach can be applied individually or in combination with other
found that the different color solutions have different effect on
nutrient stress. However, microalgae have shown varying sensitivity
biomass growth and lipid accumulation. They observed maximum
towards the surfactants and in higher concentrations it could be toxic
biomass production (1.7 gL 1) with red paint and maximum lipid
for microalgal growth [91].
content (30%) with blue paint solution. Use of LED and fluor-
ophores for improvingmicroalgal growth have shown promising
results, however, these strategies needs to be investigated further 4. Molecular approaches for enhancing lipid accumulation
including their feasibility for large scale cultivation. and quality
3.8. Chemical additives Advancements in molecular biology in the recent past, has led to
the speculation that if the genomes of oleaginous microalgae are
3.8.1. Azide appropriately modified, it is likely to have a greater impact on
Azide is known to induce oxidative stress in plants by inhibit- improving the economics of biofuel production [92–95]. Various
ing various metabolic pathways such as respiration, oxidative molecular strategies have been studied to enhance the lipid yield of
evolution of PS II and ATP-synthase. In recent investigations, azide microalgae as well as improvement in its quality of the lipids. Some
stress is reported to induce lipid accumulation in microalgae. of these recent strategies include engineering of fatty acid synthesis
Zalogin and Pick [86] compared the effect of sodium azide and pathway in microalgae towards more appropriate lipid profiles,
nitrogen stress on growth and lipid accumulation of Chlorella secretion of lipids from cells to the media, engineering of the
desiccata. Maximum lipid accumulation (60–70%) was obtained Kennedy pathway for over-expression of the major enzymes
with the addition of 20 mM sodium azide stress with minor involved in the biosynthesis of TAG, increasing the availability of
reduction in growth. They suggested azide treatment is better than
precursor molecules and inhibition of competing pathways of lipid
nitrogen deprivation as high lipid can obtain with high biomass
biosynthesis and catabolism [96]. Adaptability of microalgal strains
production. Mechanism of azide stress on lipid induction without
towards genetic manipulation is crucial for lipid enhancement
compromising growth in microalgae was explained by Zalogin and
studies. Suitability of a strain for genetic manipulation is deter-
Pick [87]. Sodium azide inhibits nitrogen assimilation by reducing
mined by various factors such as; ease of application, transforma-
activity of nitrogen reductase enzyme and results in high lipid
tion capabilities and availability of fully sequenced genomes [47].
accumulation. However, optimum azide concentration required to
enhance lipid accumulation is strain specific, which might affect
the economics of biodiesel production. More research is therefore 4.1. Microalgal genome and tools for genetic transformations in
required to know the exact mechanism behind lipid accumulation microalgae
by azide treatment, economic feasibility analysis for large scale
production and to establish a universal strategy for all microalgal Most of the previous genetic modification studies on micro-
strains. algae were based on Chlamydomonas genome as little information
was available for microalgae [19,97]. However, at present whole
3.8.2. Brefeldin A (BFA) genome sequence for many microalgaeincluding some of the
Brefeldin A is a chemical that induces stress in endoplasmic biodiesel feedstock strains are available in public domain which
reticulum (ER) and interrupt transportation of newly synthesized includes Chlorella vulgaris, Nannochloropsis, Phaeodactylum tri-
molecules (protein and lipid) from ER to golgibody and thus cornutum, Coccomyxa sp. C-169, Micromonas CCMP 1545 Ostreo-
accumulates lipids and proteins [88]. Brefeldin A is reported to coccuslucimarinus CCE9901, Ostreococcustaur, Volvox carteri, and
induce lipid droplets in Saccharomyces scerevisiae [89]. Kim et al. Thalassiosira pseudonana [19,97]. Genomic information of these
[88] has reported Brefeldin A induced an increase of 34% lipid in sequences might facilities the scope of genetic improvement of
microalgae, Chlamydomonas reinhardtii with concentration of strain by transformation. This information could open new paths
75 mgML 1. They also observed up-regulation of ER stress marker for improved genetic engineering studies in microalgae as evi-
gene and reduction of cytosolic lipid (betaine lipid diacylglyceryl denced in recent literatures
Insertion offoreign DNA molecule/s into the host cell (chlor- 4.2. Genetic engineering
oplast/mitochondria/nuclear genome) and maintaining its viability
are the most important steps for a successful transformation. Sev- 4.2.1. Chloroplast engineering
eral methods have been developed to transfer particulargenes of Microalgal chloroplast is an attractive platform for routine
interest into the microalgae. The first successful transformation was genetic manipulation as the genetic system is well suited to tar-
achieved in Chlamydomonasreihardtii by using glass beads where geted insertion into the genome that results in high level
expression of foreign genes [97,115]. In microalgae, this organelle
the cell wall free Chlamydomonasreihardtiiwas agitated in the pre-
is the site for major biosynthetic pathways including fatty acid
sence of DNA, glass beads and polyethylene glycol (PEG) [98]. This
biosynthesis, photosynthesis, xanthophylls cycle etc which can be
method is further successfully applied to transform genes in few
targeted and manipulated to enhance lipid, biomass and pigments
other microalgal strains viz, Amphidium, Sybiodium [99]. However,
production [94]. Chloroplast transformation have been success-
requirement of cell wall deficient host strain is the main drawback fully done in several high lipid producing microalgal strains such
of this method as most of the hyper lipid containing microalgal as Chlamydomonas reinhardtii, Haematococcus pluvialis [116],
strains such as Chlorella, Scenedesmus etc. are reported to have thick Dunaliella sp. [106], and Scenedesmus sp. [112]. Genetic engineer-
and complicated cell wall structure [100,101]. A more applied ing of chloroplast have resulted in high lipid accumulation in
electroporation technique was successfully employed by different transformed strain, however, decreased cell growth was reported
authors for transformation studies in various microalgae viz., in engineered strains [115,117–119]. Wobbe and Remacle [4] have
Chlamydomonasreihardtii, Chlorella eliopdodeia, Chlorella vulgaris observed improved photosynthetic efficiency by alteration in
[102], Chlorella sp. MACC/C95 [103], Phaeodactylum [104], Dunaliella light-harvesting complexes (LHC) in Chlamydomonas reinhard-
salina [105] and Nannochloropsis oculata [106]. This technique tiiusing RNAi technology. In their study, the transformed stains
involves application of electric current to disrupt the lipid bilayers showed reduction in the risk of photo inhibition thereby resulting
in the cell wall structure leading to an efficient molecular transport in improved biomass yields.Lipid accumulation and biomass pro-
duction both can be enhanced simultaneously by chloroplast
across the plasma membrane. Ahmad et al. [107] successfully used
engineering which will increase the overall volumetric lipid pro-
electroporation method to improve lipid accumulation C. reinhardtii
ductivity for economical biodiesel production.
(CC-125) where they have transformed diacylglycerol aceyl-
transferease (BnDGAT2) gene from Brassica in to Chlamydomonas
4.2.2. Nuclear engineering
reinhardtii. The engineered strain, Chlamydomonas reinhardtii Nucleus is the most attractive platform for genetic manipula-
showed higher lipid content (18.76%) than the wild strains (12.33%). tions of microalgal strain to improve lipid accumulation and
Efficiency of the technique however depends on many factors quality of biodiesel in microalgae. The whole genome analyses of
including field strength, pulse length, medium composition, tem- microalgal strains have provided substantial evidence of presence
perature and membrane characteristics as well as the concentration of lipid biosynthesis genes in different cell organelles [120,121]. It
of DNA [108]. is now known that the microalgal nuclear genome contains
Among the different techniques developed, bombardment with around 6% of total genes involved in lipid biosynthesis [121]. Most
micro-particles loaded with genetic material is the preferred of these genes are related to TAG synthesis (DGAT), fatty acid chain
method for chloroplast and nuclear genome transformation as it termination, membrane lipid synthesis and therefore alteration of
allows for the delivery of multiple copies of recombinant DNA these genes could improve both quantity and quality of the
through both the cellular and chloroplast membranes, escalating microalgal lipids in engineered strains. Advancements in bio-
the chance for a successful mixing regime to occur [105]. This technological tools, knowledge on suitable markers and promoters
facilitated microalgal nuclear transformations to a greater extent.
method can thus be applied directly to transform metabolic
Nuclear transformations in microalgae have been successfully
pathways regulated by chloroplast and nucleus such as fatty acid
carried out in several high lipid accumulating microalgal strains
biosynthesis and TAG synthesis and is successfully applied for
such as Phaeodactylum tricornutum, Nannochloropsis oceanica
stable nuclear and chloroplast transformation of C. reinhardtii,
CCMP1779 and Fistulifera sp. thus far [109,113,122].
Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella kessleri and Most success stories of microalgal nuclear transformations are
diatom Phaeodactylum tricornutum [104]. These microalgal strains based on single gene insertion. Recently, Noor-Mohammadi et al.
are already known for their high lipid content which can be fur- [123] reported a new method for multi-gene expression in nuclear
ther enhance by using this transformation method. Bombardment genome of Chlamydomonas reinhardtii. In their study, they have
is even effective for transformation of algal strains with complex developed a new method for construction of multiple-gene path-
cell wall structure such as Gonium pectoral [106] which makes this way in Saccharomyces cerevisiae and integrated this to the nuclear
technique suitable for transformations related to lipid enhance- genome of Chlamydomonas reinhardtii.Using this method, they
ment. In another study, Muto et al. [109] established a transfor- were successful in co-expressing three reporter proteins (Ble,
mation system for high lipid producing microalgae Fistulifera sp. AphVIII, and GFP) concurrently in the nucleus of C. reinhardtii. The
(61%) using microparticle bombardment methods. multi-gene expression studies has tremendous potential in GE of
Agrobacterium tumefaciens mediated transformation is the most microalgae and can be applied to improve both quantity of TAG
widely used technique for plants [108,110] due its natural ability to synthesis and quality of fatty acid synthesis simultaneously which
will have greater impact on the lipid productivity and fuel prop-
transfer inter-kingdom DNA transfer. This method can be utilized
erties of microalgal biodiesel. The above technique may also be
to improve lipid content in microalgae by expressing exogenous
utilized to express more functional genes of lipid synthesis path-
genes related to lipid metabolism. Cheng et al. [110] transformed
way as well as biomass generation to improve lipid productivity
(gfp gene) Schizochytrium using Agrobacterium tumefaciens trans-
(Table 3).
formation method; the transformed strains have shown similar
lipid and biomass content as wild strain. 4.3. Expression analysis of genes involved in lipid biosynthesis
Though there have been substantial developments with respect
to the transformation studies in microalgae, the challenge being Lipid biosynthesis pathway in microalgae has been explained by
the approach is not uniform for all algal strains [111]. several researchers [27,124,125]. Lipid biosynthesis is a multistep
reaction, catalyzed by an enzyme complex (acyl carrier protein, accumulation in the microalgae in response to cultivation condi-
fatty acid synthase) [124]. It is important to understand the tions [72,127]. Recently evolved molecular techniques such as
mechanism and behavior of these enzymes under different envir- transcriptome analysis, microarray analysis and full-length or EST
onmental conditions. Under favorable (optimum nutrients and (expressed sequence tag) transcript sequencing, can generate
cultivation parameters) conditions, microalgal growth is endorsed knowledge of how lipid biosynthetic genes are expressed under
by increased translation and transcription processes which results different stress conditionsand give insight into the key genes
in high growth rates and biomass production [19,126]. Under involved in triggering lipid accumulation [15,16,129]. The gene
nutrient limited conditions the growth of microalgae is inhibited as expression analysis therefore could assist us in improving the
most of the anabolic machinery is generally retarded. Fan et al. existing stress strategies and also to develop novel strategies for
[127] investigated the effect of nutrient stress (phosphorus, iron, better lipid yields in microalgae. Gene expression analysis also
nitrogen) on the expression of various functional genes encoding reveals the key functional genes involved in lipid biosynthesis and
the key enzymes involved in lipid synthesis of Chlorella pyrenoidosa this knowledge could be exploited for the improved applicability of
(Table 4). Similarly, Wan et al. [128] investigated the effect of iron molecular techniques such as metabolic and genetic engineering.
on lipid accumulation in Chlorella sorokinianaand observed that the
expression of genes such as accD and rbcl was up regulated at 4.4. Metabolic engineering
higher concentrations of iron resulting in high lipid productivity
(179 mgL 1). Table 4 represents the effect of environmental con- 4.4.1. Overexpression of enzymes of lipid biosynthesis
ditions on the expression of functional genes related to lipid bio- Lipid metabolism involves (Fatty acid and TAG biosynthesis) several
synthesis. These expression analyses of the key metabolic genes chemical conversion steps catalyzed by various enzymes. Acetyl-CoA
may give more insight into the actual mechanism of lipid (ACCase) carboxylase is the key enzyme involved in lipid biosynthe-
sises and the most exploited enzyme for lipid enhancement studies
Table 3 both in plants and microalgae. Overexpression of ACCase is one of the
Molecular tools and techniques for transformation of microalgae. successfully implemented strategies for improved fatty acid synthesis
Microalgae Tools and Expressed gene Reference
in plants and microorganisms which can be applied for strain
technique improvement in microalgae. It was established by various authors that
with the over expression of ACCase, enhanced availability of malonyl
Chlamydomonas Bombardment atpB [108] CoA is ensured in the chloroplast for the onset of increased fatty acid
Chlamydomonas Agrobacterium [108]
biosynthesis [130–132]. Davis et al. [133] observed 6 times increase in
Sp.
Scenedesmus Agrobacterium β-glucuronidase (uidA), [112] the rate of fatty acid synthesis while co-expressing ACCase (encoded
obliquus green fluorescent pro- by accA, accB, accC, accD) and thioesterase I (encoded by the tesA
tein (gfp) gene) in E. coli strain. These results confirmed that ACCase catalyzes
hygromycin phospho- the committing step in lipid biosynthesis which is undeniably the
transferase (hpt)
Schizochytrium Agrobacterium green fluorescent protein [110]
rate-limiting step for the fatty acid biosynthesis in E. coli [133]. The
mediated (gfp) overexpression of ACCase gene has improved the lipid accumulation in
Scenedesmus Electroporation green fluorescent protein [112] Brassica napus to 5% [134].In microalgae, under certain nutrient limited
obliquus (gfp) cultivation conditions, the expression of ACCase tend to increase [127].
Nannochloropsis sp. Electroporation GUS [113]
However, it was also reported that the overexpression of ACCase gene
Phaeodactylum Biolistic Acyl-ACP thioesterases [114]
Tricornutum transformation does not reflected into higher lipid yields; such as overexpression of
acetyl-CoA carboxylase (ACCase) from Cyclotellacryptica in
Table 4
Expression analysis of the functional genes related to lipid synthesis.
Microalgae Cultivation Targeted Selected Effect on relative gene Lipid content/fatty acid content/ Reference
Condition enzymes genes expression lipid productivity
Chlorella pyrenoidosa Nitrogen Rubisco Rbcl 4 times decrease [127]

deficient
Chlorella pyrenoidosa Nitrogen PEPC pepc g6883 Slight increase 50.4 %
deficient 34.42 mgL 1 d 1
Chlorella pyrenoidosa Nitrogen PEPC pepc g8086 Slight increase
deficient
Chlorella pyrenoidosa Nitrogen Malic me g6562 4.04 folds
deficient increase
Chlorella pyrenoidosa Nitrogen ACCase accA 3.5 folds increase
deficient
Chlorella pyrenoidosa Nitrogen ACCase accD 38.9 folds increase
deficient
Chlorella pyrenoidosa Nitrogen DGAT dgat 3280 24.5 folds
deficient
Chlorella pyrenoidosa Phosphorus Malic Me 6562 8.43 19.60 mgL 1 d 1 [127]
Chlorella pyrenoidosa Heterotrophic PEPC pepc g6883 3.1 times increase [127]
Chlorella pyrenoidosa Continuous ACCase accD 2.5 34.7% [127]
Light accA folds increase
Chlorella pyrenoidosa Heterotrophy DGTA dgta g7566 47.6 [127]
to photo trophy folds increase
Chlorella sorokiniana Mixotrophic ACCase accD Increase in expression 51% [128]
level
Chlamydomonas reinhardtii Nitrogen stress DGTA DGTT1 Increase in expression Increases [26]
level in saturated fatty acids
Cyclotellacryptica and Naviculasaprophila did not show any effect on conditions. They observed no difference in photosynthetic beha-
lipid accumulation [135]. This may be due to the fact that the ACCase viour of both the mutants and the wild type and concluded that the
activity isnotrate limiting step in the lipid biosynthesis pathway. mutation only affected the division of available carbon between
Similarly, the expression of Acyl-Co: diacylglycerol acyl- metabolic pathways and not photosynthetic performance. Astarch-
transferase (DGAT), a key enzyme in the Kennedy pathway (TAG less mutant strain of Chlamydomonas showed a 10 fold increase in
biosynthesis), has been exploited for its expression studies in TAG accumulation as compared to its wild type which was achieved
many plants like Brassica napaus, Arabidopsis thaliana and Nicoti- by deactivation of ADP-glucose pyrophosphorylase that catalyses
ana tabacum [131,136]. Few other enzymes that are targeted for the committing step in the starch metabolism [146]. These findings
enhanced fatty acid and TAG synthesis in plants and microalgae supported the potential of lipid enhancement strategy by redirect-
includes fatty acid synthatase (FAS), lysophosphatidate acyl- ing carbon from starch synthesis to lipid accumulation by knocking
transferase (LPAT), acetyl-CoA synthase (ACS), malic enzyme down the key genes in lipid biosynthesis pathway. However, knock
(ME) and ATP: citrate lyase (ACL) [95,137]. The overexpression of down of important genes involved in starch synthesis may results in
malic enzyme for enhanced lipid accumulation was studied by Xue decreased growth rate which would result in low biomass pro-
et al. [138] and the transgenic microalgae Phaeodactylum tri- duction and ultimately lower lipid productivity.
cornutum showed increased lipid content (2.5 fold) and enzymatic Suppression of lipid catabolism is one of thestrategies
activity without affecting the cell growth. employed to increase the lipid accumulation in microalgae. A
Hsieh et al. [139] over expressed the functional gene Acyl-CoA: mutant strain of Thalassiosira pseudonana has shown 3.5 times
glycerol-3-phosphate aceyltransferease (GPAT), acyl-CoA:lyso- higher lipid content after alteration in the lipid catabolism by
phosphatidicaceyltransferease (LPAAT), acyl-coA:diacylglycer- knocking down the regulation of multifunctional enzymes lipase/
olaceyltransferease (DGAT) in C. MinutissimaUTEX 2219 by using phospholipase/acyl transferase [15] (Table 5).These genes in
multiple gene expression system. The highest lipid content (2- microalgae could also be targeted for manipulation for high lipid
fold) was found in transgenic microalgal strain. The multiple-gene accumulation without compromising growth.
expression can be effectively utilized to maximize lipid accumu-
lation as well as biomass generation by targeting respective genes, 4.4.3. Altering fatty acid chain length for improved lipid quality
for sustainable biodiesel production. The microalgal lipidscomposition dictates the properties of
subsequently produced biodiesel. Thus, it is important to develop
4.4.2. Blocking competitive pathways strategies to improve the quality of microalgal lipids so that bio-
Knocking down the competitive pathways such as carbohydrate diesel can meet the standard specifications [147,148]. The most
and lipid catabolism is an effective approach to improve lipid suitable fatty acids for biodiesel production are saturated and
accumulation in microalgae [132,140–142]. Carbohydrate metabo- mono-unsaturated fatty acids preferably with carbon length 12:0,
lism is most important pathway for accumulation and storageof 14:0, 16:0, 16:1, 18:0 and 18:1. Acyl-ACP thioesterases regulates the
carbon in the form of starch employed by many microalgae fatty acid chain length by releasing the fatty acid chain from fatty
[143,144]. Suppressing the starch metabolism may therefore will acid synthase [149]. Thioesterasesfrom different organisms are
results in carbon flow towards lipid biosynthesis. Breuer et al. [145] specific for particular chain lengths of fatty acids as per the
reported an increase in TAG accumulation of upto 51% in the metabolic needs of that organism. Transformation of thioesterases
starchless mutant of Scenedesmus obliquus (0.217 gmol 1) as com- genes from other organisms into microalgae, could significantly
pared to wild type (0.144 gmol 1) under similar cultivation improve the fatty acid composition to obtain the desired fuel
Table 5
Molecular approaches to enhance lipid accumulation in microalgae.
Molecular approach Microalgae Targeted trait /pathways Result Reference
Chloroplast engineering Chlamydomonas Light harvesting complex Increase in biomass productivity [4]
(RNAi technology) reinhardtii
Metabolic engineering
Overexpression of functional genes
Acylglycerol acyltransferases Chlamydomonas Kennedy pathway Increase in mRNA level (7–29.1 times) [151]
(DGAT) reinhardtii No effect on lipid accumulation
Malic enzyme (ME) Phaeodactylum tricornutum Pyruvate metabolism Increase in expression and enzyme [138]
activity
2.5 fold increase in total lipid
content
Glycerol-3-phosphate aceyl transferase (GPAT), Chlorella minutissima Kennedy 2-fold increase in lipid content [139]
Lyso phosphatidic aceyl transferase (LPAAT), Diacyl pathway
glycerol aceyl transferase
(DGAT)
Blocking competitive pathways
Starch less mutant Scenedesmus obliquus Starch metabolism 51% increase in mutant strain as [145]
compared to wild type
ADP-glucose pyrophosphorylase Chlamydomonas Starch metabolism 10 folds increase in lipid content [146]
Knockdown of enzymes Thalassiosira pseudonana Lipid catabolism 3.5 fold increase in lipid content [15]
lipase/phospholipase/acyl transferase
Alteration in fatty acid chain length
Acyl-acp thioesterases Phaeodactylum tricornutum Fatty acid chain termination Increase quantity of in the Short chain [114]
(TE) length
Lauric and Myristic acid fatty acid
Fatty acid secretion
Overproduction of free fatty acids Synechocystis sp. Fatty acid pathway Fatty acids secretion into the medium [150]
Deletion of cyanophycin synthesis gene Cyanophycin pathway
phosphotransacetylase gene deletion Increase in production of fatty acids
properties. Radakovits et al. [114] transformed two shorter chain inevitable considering theirpossible negative impact on human
length fatty acid acyl-ACP thioesterases from Cinnamomum cam- health, environment and economy. Rigorous monitoring and risk
phora and Umbellularia californic into Phaeodactylum tricornutum. assessment studies are therefore required to design the regula-
The fatty acid profile of transgenic Phaeodactylum tricornutum tions for GM microalgae. Some non GM genetic strategies used for
showed an increase in the percentage composition of lauric enhancing lipid yields does not necessarily fall under the defini-
(C12:0) and myristic (C14:0) acids (Table 5). This molecular strat- tion set by governments for GM microalgae but could have eco-
egy for improving the microalgal lipid quality has significant logical risks. This warrants need for athorough assessment of such
potential for the production of microalgal biodiesel with the type of genetic strategies for associated risks [155].
required specifications. Another major challenge is the suitability of transgenic micro-
algae for large scale cultivation. Upon exposure to large-scale
4.4.4. Lipid secretion cultivation conditions, the strains experience situations that are
Harvesting and extraction of biomass and lipids are challen- more diverse from the controlled lab conditions, which subse-
ging, energy and cost intensive steps in the microalgal biodiesel quently affect its transgenic property. Consequently, productivity
synthesis process. Genetic modification of microalgae which in outdoor cultivation, never reaches that of the optimized
enables them to secrete the lipids into the medium can positively laboratory conditions. The stability of the mutant strain can also be
influence the economics of biodiesel production. Liua et al. [150] a challenging during commercial level cultivationas the mutants
genetically modified the cyanobacteria Synechocystis sp. PCC6803 are prone to reversion [106,154]. Though the use of algal trans-
wild type (SD100) to produce and secrete fatty acids by transgenic for enhanced lipid production is beginning to be realistic due
forming it with acyl-acyl carrier protein thioeseterase gene. In the to the advancements in molecular techniques, there are major
wild type Synechocystis sp. (SD100), fatty acid secretion was lim- environmental risks that are need to be addressed prior to the use
ited to 1.87 0.06 mgL 1 whilst the secretion of 197 714 mgL 1 of these transgenicfor largescale cultivation.
was achieved in the transgenic Synechocystis sp. (SD277). Various
metabolic pathways have been altered such as deletion of com-
petitive genes to enhance fatty acid synthesis and deletion of 5. Impacts of microalgal lipids on biodiesel properties
genes related to formation of surface protein to improve secretion
of fatty acid (Table 5). This strategy could not only make the overall Biodiesel properties are principally dictatedby the lipid content
process more energy efficient but also avoids the use of toxic of the feedstock oil. For the commercial utilization of biodiesel, it
chemicals for harvesting and minimize the usage of organic sol- has to satisfy the quality specifications set by agencies like Amer-
vents used in the extraction process. Despite of several advantages, ican Society for Testing and Materials (ASTM) D 6751 (USA); DIN
low biomass production is main drawback of this technique. 51606 (Germany); European Organization (EN 14214) and India (IS
Deletion of genes related to surface protein results in cell fragility 15607) [1]. Biodiesel is mainly characterized for the ester content,
and reduced CO2 aeration that affects photosynthesis of the cell. physical properties such as density, viscosity and fuel properties
For industrial scale production,this technique needs further mod- flash point, oxidation stability, cetane number, calorific value and
ification for better fatty acid yield. others. There is very little information available on the biodiesel
derived from the microalgal lipids. Microalgal lipids constituents
4.5. Environmental risk and challenges of molecular approach include triglycerides, free fatty acids, hydrocarbons, sterols, wax,
sterol esters, alcohols etc [3]. Other components than triglyceride
Genetic engineering approaches in microalgae have tre- and free fatty acids not only interfere with the biodiesel conversion
mendous scope in enhancing lipid production; however, there process but also have influence on the fuel properties. Hydro-
may be major limitations during the scale up. The major risk carbons present in the microalgal lipids can lead to biodiesel with
related to genetically modified (GM) microalgae can be divided high cetane number, calorific value and low viscosity [1]. Balance
into two sections. The first section is related to the human health, between the saturated and unsaturated fatty acids in total lipids is
while the other is related to the environmental impacts. Biofuels important for the oxidation stability and cold flow properties of the
from microalgae are commodity products and thus require large biodiesel. Both these properties has an inverse relation, high satu-
scale cultivation; preferablyopen cultivation systems are adopted rated fatty acids favors the oxidation stability, while high unsatu-
to reduce the production cost. Therefore, unlike other industrially rated fatty acids are considered good for the excellent cold flow
important GM organisms which are cultivated in closed systems properties of the biodiesel fuel [156]. Song et al. [157] investigated
for high value products, the risk associated with GM microalgae 10 microalgal strains for their fatty acid composition and biodiesel
could directly impact human health and environment. The escape properties. Their study indicates that fuel properties like kinematic
or release of GM microalgae to the environment is of foremost viscosity, specific gravity, cetane number and higher heating value
concern as microalgae are primary producers in most of the eco- of all the 10 microalgal strains were almost complying with the
logical systems. In open cultivation systems, GM microalgae can be ASTM D6751 and EN 14214 specifications. In their study they found
released to the environment by individual cell escape via wind, that with the increase in the unsaturated fatty acids, cold flow
water, birds, animals, natural calamities or accidents. Specific properties show the improvement however, the cetane number and
impacts could be GM species dominance over native microalgae, oxidation stability decreases. Microalgae grown with the various
change in microalgal diversity in the ecosystem, distorted food lipid enhancement strategies have shown biodiesel properties
chains and biogeochemical cycles, production of toxins and aller- complying with the standards. Baky et al. [41] studied the CO2 and
gens, and horizontal GM gene transfer to wild species [47,152]. iron supplementation for lipid enhancement in Scenedesmus obli-
Some of the microalgae are consumed as human food supplement, quus and investigated the biodiesel properties of the final biodiesel.
while some are used as main feed by fish in natural environment In their study they reported that 12% CO2 resulted in saturated fatty
[153]. Release of GM microalgae can therefore cause harm to acid composition of 59.01% and unsaturated fatty acid composition
human health as well as the environment; however, there is no of 40.99% while the degree of unsaturation was 0.52. They found
substantial data available on the magnitude of the risks and their that the density (kgm3, at 15 °C), viscosity (mm2s 1, at 40 °C), acid
impacts [154]. GM microalgae related studies are still in its early value (mg KOH g 1) and iodine value (mg I2 100 g 1) for the bio-
stages for its commercial release into outdoor cultivation sys- diesel obtained from Scenedesmus obliquus grown with 12% CO2
temsuggested that government regulations on GM microalgae are were 0.894, 4.56, 0.41 and 67 respectively. With iron
supplementation of 20 mg L 1, the saturated fatty acid composition biodiesel from chlorella protothecoides was 41 MJ kg 1 [158]and
was 57.17% and unsaturated fatty acid composition was 42.56% 40 MJ kg 1for the biodiesel from fresh water microalgae [159].
while degree of unsaturation was 0.56. The density, viscosity, acid The calorific value of microalgal biodiesel is comparable to that of
value and iodine value were 0.895, 4.53, 0.4 and 69 respectively of thediesel fuel (40–45 MJ kg 1) [158]. Overall microalgal biodiesel
biodiesel obtained from Scenedesmus obliquus grown with has shown close compliance with most of the international spe-
20 mg L 1 iron supplementation. In both the cases the biodiesel cifications, which is possible because of suitable lipid profile of
properties were comparable to the properties of the diesel fuel and microalgae. However, a very few studies have reported the prop-
also met the specifications set by ASTM (ASTM D6751) (Table 6). erties of microalgal biodiesel, indicating need of further explora-
Under the stress conditions that are mainly applied for lipid tion of this area. While applying various lipid enhancement stra-
enhancement in microalgae, unsaturated fatty acids tends to tegies it becomes imperative to investigate the quality of the lipids
undergo oxidative cleavage, which results in higher saturated fatty so that final product i.e. biodiesel meets the specifications of
acids in the microalgal lipids. Higher saturated fatty acids give standards.
oxidative stability, high heating values and cetane number. In a
study by Singh et al. [11] of using combined stress condition of
nutrients (N, P and Fe) for enhancing lipid accumulation in A. fal- 6. Conclusion and future perspective
catus the composition of unsaturated fatty acids was 76.2% without
any stress condition, and was decreased to 67.04% under stress This manuscript distinguishes itself from other reviews in the
conditions. They also observed increase in saturated fatty acid fields since it encompasses the most recent advances in lipid
composition (32.96%) under stress conditions as compared to enhancement strategies including molecular approach. To cater
unstressed condition (23.8%). the need of renewable transportation fuels colossal amount of
Microalgal lipids have shown C12–C18 fatty acids as the major biodiesel is required. Economical production of biodiesel from
contributing fatty acids, these fatty acids are considered suitable microalgae can be achieved by ensuring high lipid accumulation in
for the biodiesel synthesis (Table 7). C12–C18 fatty acids have the microalgal cells. Conventional approaches such as altering nutrient
viscosity in the range of 2.43–5.85 mm2s 1, and thus the resulting regime and cultivation conditions are either expensive or asso-
biodiesel also has lower viscosity values and satisfy the ASTM ciated with overall low lipid productivity. To address these chal-
D6751 specified range of 3.5–5 mm2s 1. The calorific value of lenges, several novel approaches have been investigated in the
Table 6
Fuel properties of microalgal biodiesel.
Biodiesel Cetane Calorific Density Methyl Linolenic acid Acid value Iodine Cold filter oxidative Sulfur Ref
characteristics number value ester methyl ester number plugging stability
content content point (CFPP)
Units – MJ kg 1 kg m 3 % % mgKOH g 1 g100 g 1 °C h wt% –

ASTM 6751 min 47 – 860–900 – – max 0.5 - – min 3 max 0.05 –
EN 14214 min 51 – 860–900 min 96.5 max 12 max 0.5 max 120 – min 6 – –
Chlorella – 41 864 – – 0.374 – 11 – – [160]
protothecoides
Scenedesmus 51.77 37.67 877 90.81 11.17 0.42 98.68 4.9 3.54 o 0.001 [161]
obliquus
Nannochloropsis sp. – – 854 92.2 – 0.46 – – 1.53 0.06 [162]
Spirulina platensis 70 45.63 863 – – 0.75 102 – – nil [163]
Chlorella – – 882 97.7 – 0.29 112.2 13 4.52 – [162]
protothecoides
Table 7
Lipid content and fatty acid composition of different microalgae.
Microalgal strain Lipid content (%) Fatty acid composition (%) Ref.
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 SFA MUFA PUFA
Chlorella vulgaris 17.3 – 14.55 1.18 10.51 23.62 13.8 32.1 25.06 24.8 45.9 [164]
Scenedesmus sp. 30–36 – 18.42 2.31 3.43 49.64 11.30 8.26 21.85 51.95 22.82 [162]
Chlorella sp., BUM11008 31.2 0.3 0.41 2.41 25.94 6.85 16.17 0.99 68.21 12.03 19.76 [31]
Dunaliella sp. 22 – 9.19 0.8 4.27 22.51 3.84 44.31 13.47 24.74 48.15 [164]
Nannochloropsis sp. – 5.37 28.83 32.93 0.98 21.16 2.24 – 35.18 54.09 8.57 [162]
Chlamydomonas reinhardtii 18.9 – 23.77 1.94 4.41 19.73 6.58 25.49 18.18 22.88 32.07 [164]
Dianoflagellate -– 6.01 16.65 3.35 – 2.10 – – 25.31 5.45 66.14 [162]
Chlorococcum sp 7.1 – 19 4 3 63 4 – – – – [165]
Monoraphidium sp. FXY-10 56.80 – 22.6 – 1.2 – 4.2 42.2 23.8 – 68 [166]
Neochloris oleoabundans 10–15 – 23–30 0.6–3.5 0.9–11 30–43 18–23 5–12 – – – [167]
Selenastrum capricornutum 27.08 – 45.62 5.96 – 4.27 11.75 – E48 E10 E17 [157]
Phaeodactylum tricornutum 61.43 11.68 0.13 22.34 1.49 7.42 0.81 0.25 E15 E30 E42 [157]
Isochrysis sphacrica 24.28 28.17 39.39 – – 22.26 – – E67 E21 – [157]
Chlorella sp. 12 2.12 16.36 6.09 1.22 33.69 11.96 12.74 19.7 39.9 37.47 [168]
Nannochloris sp. 21 2.03 25.28 2.36 0.98 5.83 19.65 23.24 28.29 8.19 49.5 [168]
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Trends and Novel Strategies For Enhancing Lipid Accumulation and Quality in Microalgae

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Trends and novel strategies for enhancing lipid

Article in Renewable and Sustainable Energy Reviews · November 2015

Sheena Kumari Rohit Misra

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Ismail Rawat Faizal Bux

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Cultivation of microalgae in open raceway system using wastewater at demonstration scale

Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews

Trends and novel strategies for enhancing lipid accumulation

4.3. Expression analysis of genes involved in lipid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1. Introduction 2.1. Effect of nutrient regimes on lipid accumulation

2. Nutrient regime and cultivation conditions to enhance lipid

Microalgae are able to survive in extreme environments as they

Fig. 2. Molecular strategies for enhancing lipid accumulation in microalgae.

Strategies Advantages Challenges

3.1. Two-stage cultivation 3.2. Combined nutrients and abiotic stress

3.3. Phytohormones 3.5. Co-cultivation of microalgae–bacteria

Chlorella pyrenoidosa Nitrogen Rubisco Rbcl 4 times decrease [127]

Molecular approach Microalgae Targeted trait /pathways Result Reference

Units – MJ kg 1 kg m 3 % % mgKOH g 1 g100 g 1 °C h wt% –

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