Table 9 Colorimetric methods for lipid quantification of oleaginous microorganisms

Copper extraction in chloroform

Centrifuge cells and freeze pellet, thaw and saponify pellet (with 25% methanol in 1 N NaOH), add 59 mg glass beads for cell disruption, heat at 100 °C for 30 min, add 200 μL neutralization reagent and 200 μL copper reagent, add 250 μL chloroform and centrifuge, add 50 μL of organic phase and 50 μL sodium diethyldithiocarbamate in 2-butanol, measure absorbance at 440 nm

Phaeodactylum tricornutum, Chlorella vulgaris

0.02–0.8 μmol fatty acid

Small amout of culture (1–2 mL culture); adaptation to microcentrifuge format allows up to 30 samples in < 2 h; requires cell disruption

Laurate, decanoate

–

[192]

SPV, modified for microplate high-throughput screening

Evaporate solvent, add 100 μL sulfuric acid, and incubate at 90 °C for 20 min, cool microplate for 2 min on ice, add 50 μL vanillin-phosphoric acid, wait 10 min, measure absorbance at 540 nm

4 Chlorella species

5–120 μg

Small amount of sample (< 100 μL), less time (< 1 h), color development is more consistent due to modification of reagent concentrations; requires extraction and purification

Corn oil

Gravimetric

[193]

SPV

Prepare sample (extraction), evaporate solvent, add 100 μL sample, add 2 mL sulfuric acid, heat at 100 °C for 10 min, cool for 5 min in ice bath, add 5 mL vanillin-phosphoric acid, incubate at 37 °C for 15 min at 200 rpm, measure absorbance at 530 nm

Chlorella sp., Monoraphidium sp., Ettlia sp., Nannocloropsis sp.

100–1000 mg

Applicable to wide range of microalgae

Canola oil

GC/MS

[194]

Automated quantification on spinning disc with TAG colorimetric detection based on a commercial kit

Automated process

Chlamydomonas reinhardtii

5–30 μg

Includes automated cell sedimentation lysis and extraction; reduced time (< 13 min); only TAGs; small amount of sample (≤500 μL)

Triglyceride lipid standard

GC/MS, gravimetric

[195]

SPV

Add 0.2–1 mg dried biomass in distilled water, incubate at 100 °C for 10 min, cool for 5 min in ice bath, add 5 mL vanillin-phosphoric acid, incubate at 200 rpm and 37 °C for 15 min, measure absorbance at 530 nm

Schizochytrium sp., Thraustochytrium sp.,

0.2–1 mg

No solvent addition, applied to whole cells; reduced time (30 min)

Commercial Schizochytrium oil

Gravimetric

[191]

SPV, modified for microplate high-throughput screening

Prepare sample, dissolve 20 μL sample in 180 μL concentrated H₂SO₄, heat at 100 °C for 10 min and cool to room temperature, add 0.5 mL vanillin-phosphoric acid, heat at 37 °C for 15 min and cool to room temperature, store for 45 min in dark box, measure absorbance at 530 nm

C. vulgaris, Lipomyces starkeyi, fresh and lyophilized

up to 20 mg/mL

Small amount of sample (20 μL); no solvent addition as preliminary step; reduced time (< 1 h)

Oleic acid, palmitic acid

Gravimetric

[196]