Green Solvents and Ball Mills Team Up to Pull Valuable Lutein from Microalgae

Lutein, the golden-yellow pigment that accumulates in the human retina and shields our eyes from damaging light, has long been harvested the hard way: from marigold petals grown seasonally, picked and separated by hand. Now researchers have unveiled a greener, faster route to this prized carotenoid, one that starts not in a flower field but in a vat of microscopic algae, and relies on solvents whose ingredients could plausibly come from a kitchen pantry. A team led by Gul Muhammad reports in the journal Results in Chemistry a ball milling-assisted extraction method that uses natural deep eutectic solvents, or NADESs, to recover lutein from Chlorella pyrenoidosa more efficiently, more safely, and with far less environmental baggage than conventional organic solvents.

The stakes are considerable. Lutein is the second most valuable carotenoid produced from microalgae after astaxanthin, feeding a global market currently worth about 309 million US dollars and projected to reach 406 million by 2026. Beyond supplements and food additives, the pigment’s potent antioxidant activity has made it a fixture of research into human eye health. Traditional extraction, however, leans on solvents such as tetrahydrofuran, acetone, and diethyl ether, all of which are flammable, volatile, and raise toxicity concerns. Supercritical carbon dioxide offers an alternative but demands heavy energy inputs, while ionic liquids, though effective, are costly to synthesize and not readily biodegradable, hampering their scale-up.

NADESs occupy a sweet spot between performance and sustainability. These solvents form when a hydrogen bond acceptor, such as the plant-derived compound thymol, is mixed with a hydrogen bond donor, such as decanoic acid or isoborneol, and gently heated. The components melt together into a liquid whose hydrogen bonding network gives it unusual solvation powers. Unlike conventional solvents, they require no purification step, generate no synthetic waste, and break down readily in the environment. Crucially for carotenoid work, many NADESs also suppress oxidative degradation, meaning the pigment they extract tends to survive longer.

In the new study, the researchers synthesized six different NADES formulations and characterized them thoroughly using infrared spectroscopy, viscometry, and density measurements. The telltale signature of successful eutectic formation appeared in the spectra: the hydroxyl stretching band of thymol shifted from roughly 3175 to 3418 inverse centimeters, and the carbonyl band of decanoic acid moved to higher wavenumbers, confirming that hydrogen bonds had stitched the components into a new liquid. Densities fell between 0.90 and 0.94 grams per cubic centimeter, while viscosities spanned a wide range, from a pourable 11.67 to a sluggish 98.40 millipascal-seconds at room temperature, a property the team knew would matter enormously for mass transfer during extraction.

The real innovation, however, came before any solvent touched the algae. Chlorella cells are wrapped in tough cell walls that resist solvent penetration, so the team pretreated the biomass in a planetary ball mill, essentially a jar of grinding beads that pulverizes the cells through high-speed collisions. Optimizing three variables proved decisive. Milling for 30 minutes at 400 revolutions per minute with a biomass-to-beads ratio of 1:30 lifted the lutein yield from 0.61 to 2.02 milligrams per gram of dry biomass. Pushing the speed to 500 rpm actually backfired: centrifugal forces compacted the biomass into a cake layer on the jar walls and balls, wasting energy and partially degrading the pigment. Scanning electron microscopy revealed the physical aftermath, with pretreated cells visibly ruptured and shrunken, and surface area measurements confirmed the effect, rising from 4.13 to 7.30 square meters per gram after milling.

When the milled biomass met the solvent candidates, one formulation stood out. A 1:1 mixture of thymol and isoborneol, designated N3, outperformed all five competitors and beat the conventional solvent ethyl acetate, which yielded only 1.62 milligrams per gram. The researchers note that these particular thymol and menthol-based eutectic mixtures had never previously been applied to lutein recovery from microalgae. The chemistry of the extracts told its own story: acid-containing NADESs produced yellowish extracts as acidic conditions converted chlorophylls into pheophytins, stripping magnesium from the porphyrin ring, while the non-acidic N3 yielded the green tinge characteristic of intact chlorophyll co-extraction.

Fine-tuning the extraction itself pushed yields higher still. A solid-to-liquid ratio of 0.067 grams per milliliter proved optimal, delivering 33.86 percent more lutein than a leaner ratio, while diluting the NADES with ethanol to a 50:50 blend cut viscosity dramatically, from the neat solvent down to under 2 millipascal-seconds in some dilutions, and boosted recovery to 2.34 milligrams per gram. A single extraction cycle sufficed to capture most of the pigment, a direct payoff of the ball milling pretreatment, though additional cycles squeezed out further gains.

Perhaps the most forward-looking result concerns solvent recycling, a persistent headache for eutectic solvent chemistry because the strong molecular interactions that make NADESs good extractants also make them hard to separate from the extracted compounds. Using a graphitized carbon black cartridge in a solid-phase extraction setup, the team demonstrated that the NADES-ethanol blend retained 88.40 percent of its original extraction efficiency after one recycling cycle, 82.46 percent after two, and still a workable 70.89 percent after three. The trapped lutein itself could be eluted from the cartridge with 2-methyltetrahydrofuran, recovered at 56.41 percent efficiency, meaning neither the pigment nor the solvent is simply thrown away.

The NADES system also proved a better bodyguard for the delicate pigment. Lutein degrades under heat, light, and oxygen, and isomerizes into less useful forms when overheated. In thermal challenge tests at 40, 60, and 80 degrees Celsius, lutein dissolved in the NADES-ethanol blend degraded just 2.53 percent in the first half hour at 40 degrees, compared with 7.05 percent in methanol, and after 150 minutes the NADES sample still held 85.1 percent of its lutein versus 81.02 percent for the methanol control. Half-life calculations underscored the difference: 608 minutes at 40 degrees Celsius in the NADES blend against 450 minutes in methanol. Thermodynamic analysis showed the degradation required more activation energy in the NADES system, 18.73 versus 11.79 kilojoules per mole, meaning the solvent imposes a higher energy barrier against breakdown, and positive Gibbs free energy values confirmed the pigment does not degrade spontaneously in either medium.

A formal sustainability audit sealed the comparison. Using the Green Analytical Procedure Index, which scores every stage of a method from reagents to waste disposal with color-coded pictograms, the NADES-based process earned six green, seven yellow, and two red ratings, while a traditional organic solvent extraction scored three green, eight yellow, and four red. The gap stemmed chiefly from safer extractants and recyclable solvent handling. Taken together, the findings sketch a pipeline in which rapidly grown, carbon-sequestering microalgae are shattered by precisely tuned mechanical forces, their lutein dissolved in plant-derived liquids that can be reused repeatedly, and the pigment protected all the way to the bottle. For a market hungry for natural antioxidants, that combination of efficiency, mildness, and green credentials may be exactly what sustainable carotenoid production has been waiting for.

Subject of Research: Sustainable extraction of the carotenoid lutein from microalgae using ball milling and natural deep eutectic solvents

Article Title: Efficient ball milling assisted approach to recover lutein from microalgae biomass with natural deep eutectic solvents

Article References: Muhammad, G., Yuan, X., Li, Z., Zhang, X., & Zhang, Z. (2026). Efficient ball milling assisted approach to recover lutein from microalgae biomass with natural deep eutectic solvents. Results in Chemistry, 30, Article 103861. https://doi.org/10.1016/j.rechem.2026.103861

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103861

Keywords: lutein, microalgae, Chlorella pyrenoidosa, natural deep eutectic solvents, ball milling, green extraction, carotenoids, antioxidants, solvent recycling, thermal stability, biorefinery, sustainability