Table 1 – Manipulation of environmental conditions and genetic engineering attempts to increase lipid production and biomass accumulation in diatoms



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Table 1 – Manipulation of environmental conditions and genetic engineering attempts to increase lipid production and biomass accumulation in diatoms.

EASW, enhanced artificial sea water; PUFA, polyunsaturated fatty acids; MUFA, monounsaturated fatty acids; SFA, saturated fatty acids; TFA, total fatty acids.




Diatom

Stress conditions / transformation / mutagen

Growth conditions

Growth rates

(day -1)

Physiological changes

Reference

Nitrogen limitation/starvation

Phaeodactylum tricornutum UTEX640

(Marine)


Nitrogen limitation (From 2 to 10 mM)

Fermenter, EASW, 1200 μmol photons m2 s-1, 20 °C

0 .29-0.57

Total lipids were 11% of the dry weight.

For cultures grown at 0.29 d-1, the lipid content increased to 19% upon nitrogen starvation.

TAG levels increased from 69 to 75% of the total lipid content with nitrogen limitation.


[1]

Phaeodactylum tricornutum

Nitrogen starvation

ASW based F/2, 300 μmol photons m2 s-1 white fluorescent, continuous light, 18 °C

0.97 (+N);

0.21 [2]


TFA increased by 76%, from 2.39 to 4.21 pg cell-1
Quantum requirement for incorporating FA, increased by 216%

[3]

Phaeodactylum tricornutum

Nitrogen starvation

ASW based F/2, 200 μmol photons m2 s-1 white LED, continuous light, 18 °C

0.86 (+N);

0.31 [2]


TFA increased by 21%, from 2.9 to 3.5 pg cell-1
Quantum requirement for incorporating FA, increased by 155%

[4]

Thalassiosira pseudonana (CCMP 1335)

(Marine)



Nitrogen starvation

Natural seawater, 210 μmol photons m2 s-1 12:12h L:D, 19 °C.

1.21 (+N);

0 [2]


Lipid content increased from 13% to 20% of the dry weight

[5]

Chaetoceros muelleri (Schütt)

(Marine)


Nitrogen starvation

SERI Types I and II media, 150 μmol photons m2 s-1, 5 - 45 °C.

Growth rates average ~1.5 d-1 and reached as high as 3.5 d-1 at high temperature (30 °C) under N replete conditions

Total lipid content increased by 7-folds after nitrogen starvation.


[6]

Navicula pelliculosa
(Freshwater)

Nitrogen starvation (7-9 days)

WC medium 23 °C, 190 µmol photons m-2 s-1

1.1 (during exponential phase)

Total lipids content reached 44.8% of the cell’s dry weigh.

[7]

Nitzschia palea

(Freshwater)



Nitrogen starvation

(7-9 days)



WC medium 23 °C, 190 µmol photons m-2 s-1

3.8 (during exponential phase)

Total lipids content increased from 22 to 40% of the cell’s dry weight.

[7]

Cyclotella cryptica
(Marine)

Nitrogen starvation

(7-9 days)



F/2 medium 23 °C, 190 µmol photons m-2 s-1

2.6 (during exponential phase)

Total lipids content increased from 23 to 37% of the cell’s dry weight.

[7]

Skeletonema costatum
(Marine)

Nitrogen starvation

(7-9 days)



F/2 medium 23 °C, 190 µmol photons m-2 s-1

2.7 (during exponential phase)

Total lipids content increased from 24 to 30% of the cell’s dry weight.

[7]

Silicates limitation/starvation

Thalassiosira pseudonana

(Marine)


Silicate starvation

n/a

n/a

Increased in natural lipid abundance occurred faster and to a higher extent than when starving the cells to nitrogen.

[8]

Chaetoceros gracilis

(Marine)

Silicate starvation

(7.6 – 30 µM Si)



FSW enriched with nutrients; 12:12 light:dark cycle, 100 µmol photons m-2 s-1; 25 °C. Initial Si concentrations 2-50 µM

1.3 ± 0.04 for all Si concentrations

A maximum of 46% lipids of dry weight.
Value is reported by re calculating lipid carbon contents using biochemical compulsion calculation.

[9]

Cyclotella cryptica

(Marine)


Silicate starvation

85 μmol photons m2 s-1, 1 % CO2, 23 – 25 °C

1.24 (normal)

0.62 (silicon deficient)



Increased lipid content from 20% to 28% of dry weight after 12 h of silicon deficiency

[10]

Cyclotella sp.

(Marine)

Silicate starvation

(3.1-49.3 µM Si)



FSW enriched with nutrients; 12:12 light:dark cycle, 100 µmol photons m-2 s-1; 25 °C. Initial Si concentrations 2-50 µM

1.4 ± 0.08 for all Si concentrations

A maximum of 61% lipids of dry weight.
Value is reported by re calculating lipid carbon contents using biochemical compulsion calculation.

[9]

Hantzschia sp.

(Marine)

Silicate starvation

(2.1-45.7 µM Si)



FSW enriched with nutrients; 12:12 light:dark cycle, 100 µmol photons m-2 s-1; 25 °C. Initial Si concentrations 2-50 µM

1.6 ± 0.08 for all Si concentrations

A maximum of 54% lipids of dry weight.
Value is reported by re calculating lipid carbon contents using biochemical compulsion calculation.

[9]

Navicula pseudotenelloides

Silicate starvation

Artificial seawater and an array of SERI media and conditions

2.5 at 30 25 °C

43% lipids of dry weight under Si starvation.

[11]

Navicula acceptate

Silicate starvation

Artificial seawater and an array of SERI media and conditions

1.5-3

47% lipids of dry weight sunder Si starvation.

[11]

Navicula pelliculosa
(Freshwater)

Silicate limitation (50 hours)

Tryptone (Difco) medium, 20 °C, and 17,000 lux (~230 µmol photons m-2 s-1).

For Si limitation - Si (8 µg mL-1),



n/a

Lipids were 25% of the dry weight during exponential phase.

Lipid content increased by 36% to reach 34% of dry weight.




[12]

Nitzschia dissipata

Silicate starvation

n/a

2.5

An average of 46% lipids of dry weight under Si starvation.

[11]

Phosphorus limitation/starvation

Phaeodactylum tricornutum

(Marine)


Phosphorus limitation

F/2 medium, 70-90 μmol photons m2 s-1, 23 – 25 °C

1.06 (normal)

0.53


(moderate limitation)

0.047 (limited)



Total lipid content increased from 8% to 16% of dry weight.
TFA constituted 41% of the total lipids.

[13]

Chaetoceros sp.

(Marine)


Phosphorus limitation

F/2 medium, 70-90 μmol photons m2 s-1, 23 – 25 °C

1.29 (normal)

0.006 (limited)



Total lipid content increased from 8% to 19% of dry weight.
Total fatty acids constituted 43-46% of the total lipids.

[13]

Combination of nutrients limitation/starvation

Stephanodiscus minutulus

(Freshwater)



Silicate, nitrogen or phosphorus limitation

COMBO medium, 80 μmol photons m-2 s-1 14:10h L:D, 16 °C.

0.17-0.84


Up to 50% increase in the percentage of lipids from dry weight. From 34% to 51% lipids from dry weight under Si limitation.

[14]

Temperature stress

Phaeodactylum tricornutum 2038 (obtained from the Institute of Oceanography, the Chinese Academy of Sciences, Qingdao)
(Marine)


Temperature stress

F/2 medium (increased 4-fold urea and 2-fold phosphate), 130 μmol photons m-2 s-1 16:8h L:D; 25, 20, 15, or

10 °C



n/a

EPA and PUFA increased by 120% after shift from 25 °C to 10 °C for 12 h.
The percentage of PUFA from total lipids increased from 22% to 32%, reaching as high as 4.9% of the total dry weight.

The percentage of EPA from total lipids increased from 13% to 24%, reaching as high as 2.6% of the total dry weight.



[15]

Chaetoceros sp.

CS256)


(Marine)


Temperature stress

F/2 medium, 80 μmol photons m-2 s-1 12:12h L:D, pH 8.3; 25,

27, 30, 33 and 35 °C.



0.74 (at 25 °C) - 0.87 (at 30 °C)


Total lipid per dry weight increased from 12 % at 35 °C to 16.8% at 25 °C

[16]

Light and UV stress

Phaeodactylum

Tricornutum

(Marine)


UV radiation

Natural seawater enriched f/2 medium, 60 μmol photons m-2 s-1 16:8h L:D, 18 °C.

1.38 (normal conditions)

Increase of PUFA and a reduction of SFA.

[17]

Thalassiosira pseudonana

(Marine)



Light and photoperiod

G2 medium, 20 °C, 1% CO2; 100 μmol photons m-2 s-1 12:12h L:D, 100 μmol photons m-2 s-1 24:0h L:D, 50 μmol photons m-2 s-1 24:0h L:D.

1.9 (100 μmol photons m2 s-1 12:12h L:D)

1.5 (100 μmol photons m2 s-1 24:0h L:D)

1.0 (50 μmol photons m2 s-1 24:0h L:D)


Total percentage of lipids from dry weight increased from 21% during logarithmic growth phase to 31% in stationary growth phase.
Under high light - Percentage of TAG from total lipid increase from 7-10% in logarithmic growth phase to 47% during stationary growth phase (100 μmol photons m2 s-1).

[18]

Chaetoceros muelleri

(Marine)


UV radiation

Natural seawater enriched f/2 medium, 60 μmol photons m-2 s-1 16:8h L:D, 18 °C.

0.8 (normal conditions)

Increase of PUFA and a reduction of SFA.

[17]

Chaetoceros simplex

(Marine)


High UV-B radiation

F/2 medium, 85 μmol photons m-2 s-1 12:12h L:D, 20 °C.

n/a

Increase of total lipid content by 67% under high UV-B treatment.

[19]

Cyclotella meneghiniana (Kütz)
(Marine)


Light intensity and phosphorus supply

WC medium; 30, 60, 140,

230, and 490 μmol photons m2 s-1; 20 °C



n/a

Higher levels of TFA, SFA, and MUFA with increasing light intensity.

TFA increased from 400 to 460 µg/mg C in the high-P cultures, and from 400 to 570 µg/mg C in the low-P treatment with light.



[20]

Nitzschia closterium

Light

Synthetic seawater,

17 °C, 1% CO2; ~30 and ~300 μmol photons m-2 s-1



n/a

TFA increase from 7.6% to 12.6% of dry weigth (66% increase).

TGAs increased from 1.7% to 9.2 of dry weight (440% increase).



[21]

Phaeocystis Antarctica

(marine)


High UV-B radiation

F/2 medium, 85 μmol photons m-2 s-1 12:12h L:D, 20 °C.

n/a

Increase of total lipid content by 1000% under high UV-B treatment.

[19]

Cell cycle inhibition

Thalassiosira pseudonana

(Marine)


Nocodazola

n/a

n/a

Increased in natural lipid abundance occurred faster and to a higher extent than when starving the cells to nitrogen.

[8]

Nitzschia sp.

(freshwater)



Using old sterile media of Nitzschia sp. With sufficient nitrogen source

80 μmol photons m-2 s-1, 30 °C, 1.5% CO2 bublling

n/a

Increase of 142% in lipid content after 1 day, and 542% of the lipid content in 4 days.

[22]

Other environmental/chemical manipulations

Phaeodactylum tricornutum

Addition of 0.88 mM sodium tungstate (without Molibdinum)

ASW based F/2, 300 μmol photons m2 s-1 white fluorescent, continuous light, 18 °C

0.97 (+N);

0.27 [2]


TFA increased by 67%, from 2.39 to 3.99 pg cell-1
Quantum requirement for incorporating FA, increased by 162%.

[3]

Phaeodactylum tricornutum

Addition of 0.88 mM sodium tungstate (without Molibdinum)

ASW based F/2, 200 μmol photons m2 s-1 white LED, continuous light, 18 °C

0.86 (+N);

0.66 [2]


TFA increased by 14%, from 2.9 to 3.3 pg cell-1
Quantum requirement for incorporating FA did not change.

[4]

Nitzschia laevis (UTEX 2047)

(Marine)


Salt stress

Modified Lewin’s medium, NaCl was adjusted to 5, 10, 20, and 30 g L-1.

From 0.58 at 10 g NaCl L-1

to 0.41 at 40 g NaCl L-1



Percentage of TAGs from total lipids increased from 37% to 69% when reducing NaCl from 30 g L-1 to 10 g L-1.
Percentage of natural lipids from total lipids was increased from 43% to 79% when reducing NaCl from 30 g L-1 to 10 g L-1.

[23]

Genetic manipulations

Cyclotella sp. wild type CYCLOJ5

(Marine)


Random mutagenesis using ethylmethylsulfonate

30 μmol photons m-2 s-1 continuous light
50% ASW



WT grow faster (3.45 d-1) then the CM1 mutant (3.3 d-1).


The light intensity at which photosynthesis saturates was 2-3 times greater in the pigment mutant CM1-1.

No improvements in biomass productivity were observed in either semi-continuous laboratory cultures or outdoor ponds.



[24]

Cyclotella cryptica T13L (Marine)


Expressing of Acetyl-CoA caboxylase (acc1) from C. cryptica T13L

50 μmol photons m-2 s-1, 16:8 dark/light
50% ASW


n/a

Transformants did not exhibit increase in oil production.

Over-expression of acc1 led to a 2-3-fold in enzyme activity



[11, 25]


Phaeodactylum tricornutum

CCMP632


(Marine)

Over expression of 2 different plant thioesterases:

myristic acid biased (C14-TE) and lauric acid biased thioesterase (C12-TE) from from Cinnamomum camphora




60 μmol photons m-2 s-1 continuous light
50% F/2- Si
22 °C


Slower growth observed in the transgenic strains

Increased accumulation of short saturated chain length fatty acids with no significant increase in secretion of fatty acids.

[26]

Thalassiosira pseudonana

(Marine)

Knockdown of a multifunctional lipase/phospholipase/acyltransferase Thaps3_264297

Continuous illumination or a 12 h:12 h light:dark cycle at 150 µmol μmol photons m-2s-1 at 1820 °C

ASW medium supplemented with biotin and vitamin B12



Antisense-expressing knockdown strains exhibited wild-typelike growth

Transformants showed 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation.

[27]


References for Table S1
1. Alonso, D.L., et al. (2000) Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum. Phytochemistry 54, 461-471

2. Rasconi, S., et al. (2011) Parasitic fungi of phytoplankton: ecological roles and implications for microbial food webs. Aquat Microb Ecol 62, 123-137

3. Frada, M.J., et al. (2013) Quantum requirements for growth and fatty acid biosynthesis in the marine diatom Phaeodactylum tricornutum (Bacillariophyceae) in nitrogen replete and limited conditions. J Phycol 49, 381-388

4. Guerra, L.T., et al. (2013) Regulatory branch points affecting protein and lipid biosynthesis in the diatom Phaeodactylum tricornutum. Biomass Bioenergy 59, 306-315

5. Jiang, Y.L., et al. (2012) Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae. Plant Physiol. Biochem. 54, 70-77

6. McGinnis, K.M., et al. (1997) Characterization of the growth and lipid content of the diatom Chaetoceros muelleri. J Appl Phycol 9, 19-24

7. Shifrin, N.S. and Chisholm, S.W. (1981) Phytoplankton lipids: interspecific differences and effects of nitrate, silicate and light-dark cycles. J Phycol 17, 374-384

8. Hildebrand, M., et al. (2012) The place of diatoms in the biofuels industry. Biofuels 3, 221-240

9. Taguchi, S., et al. (1987) Silicate deficiency and lipid-synthesis of marine diatoms. J Phycol 23, 260-267

10. Roessler, P.G. (1988) Effects of Silicon Deficiency on Lipid-Composition and Metabolism in the Diatom Cyclotella cryptica. J Phycol 24, 394-400

11. Sheehan, J., T. Dunahay, J. Benemann, and P. Roessler (1998) A look back at the US Department of Energy’s Aquatic Species Program—biodiesel from algae. National Renewable Energy Laboratory

12. Coombs, J., et al. (1967) Studies on the biochemistry and fine structure of silica shell formation in diatoms. Chemical composition of Navicula pelliculosa during silicon-starvation synchrony. Plant Physiol 42, 1601-1606

13. Reitan, K.I., et al. (1994) Effect of nutrient limitation on fatty-acid and lipid-content of marine microalgae. J Phycol 30, 972-979

14. Lynn, S.G., et al. (2000) Effect of nutrient availability on the biochemical and elemental stoichiometry in the freshwater diatom Stephanodiscus minutulus (Bacillariophyceae). J Phycol 36, 510-522

15. Jiang, H.M. and Gao, K.S. (2004) Effects of lowering temperature during culture on the production of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum (Bacillariophyceae). J Phycol 40, 651-654

16. Renaud, S.M., et al. (2002) Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 211, 195-214

17. Liang, Y., et al. (2006) Effects of nitrogen source and UV radiation on the growth, chlorophyll fluorescence and fatty acid composition of Phaeodactylum tricornutum and Chaetoceros muelleri (Bacillarlophyceae). J Photoch Photobio B 82, 161-172

18. Brown, M.R., et al. (1996) Effects of harvest stage and light on the biochemical composition of the diatom Thalassiosira pseudonana. J Phycol 32, 64-73

19. Skerratt, J.H., et al. (1998) Effect of UV-B on lipid content of three Antarctic marine phytoplankton. Phytochemistry 49, 999-1007

20. Piepho, M., et al. (2012) Species-specific variation in fatty acid concentrations of four phytoplankton species: does phosphorus supply influence the effect of light intensity or temperature? J Phycol 48, 64-73

21. Orcutt, D.M. and Patterson, G.W. (1974) Effect of light intensity upon lipid composition of Nitzschia closterium (Cylindrotheca fusiformis). Lipids 9, 1000-1003
22. Badour, S.S. and Gergis, M.S. (1965) Cell division and fat accumulation in Nitzschia sp. grown in continuously illuminated mass cultures. Arch Mikrobiol 51, 94-102

23. Chen, G.Q., et al. (2008) Salt-Induced alterations in lipid composition of diatom Nitzschia laevis (bacillariophyceae) under heterotrophic culture condition. J Phycol 44, 1309-1314

24. Huesemann, M.H., et al. (2009) Biomass Productivities in Wild Type and Pigment Mutant of Cyclotella sp. (Diatom). Appl Biochem Biotechnol 157, 507-526

25. Dunahay, T.G., et al. (1996) Manipulation of microalgal lipid production using genetic engineering. Appl Biochem Biotechnol 57-8, 223-231



26. Radakovits, R., et al. (2011) Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metab Eng 13, 89-95

27. Trentacoste, E.M., et al. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proceedings of the National Academy of Sciences of the United States of America 110 (49) 19748-19753


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