Kyubock Lee

Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus

Recently, biofuels and nutraceuticals produced from microalgae have emerged as major interests, resulting in intensive research of the microalgal biorefinery process. In this paper, recent developments in cell-wall disruption and extraction methods are reviewed, focusing on lipid and astaxanthin production from the biotechnologically important microalgae Chlorella and Haematococcus, respectively. As a common, critical bottleneck for recovery of intracellular components such as lipid and astaxanthin from these microalgae, the composition and structure of rigid, thick cell-walls were analyzed. Various chemical, physical, physico-chemical, and biological methods applied for cell-wall breakage and lipid/astaxanthin extraction from Chlorella and Haematococcus are discussed in detail and compared based on efficiency, energy consumption, type and dosage of solvent, biomass concentration and status (wet/dried), toxicity, scalability, and synergistic combinations. This report could serve as a useful guide to the implementation of practical downstream processes for recovery of valuable products from microalgae including Chlorella and Haematococcus.

Improved biomass and lipid production in a mixotrophic culture of Chlorella sp. KR-1 with addition of coal-fired flue-gas.

Industrial CO2-rich flue-gases, owing to their eco-toxicity, have yet to be practically exploited for microalgal biomass and lipid production. In this study, various autotrophic and mixotrophic culture modes for an oleaginous microalga, Chlorella sp. KR-1 were compared for the use in actual coal-fired flue-gas. Among the mixotrophic conditions tested, the fed-batch feedings of glucose and the supply of air in dark cycles showed the highest biomass (561 mg/L d) and fatty-acid methyl-ester (168 mg/L d) productivities. This growth condition also resulted in the maximal population of microalgae and the minimal population and types of KR-1-associated-bacterial species as confirmed by particle-volume-distribution and denaturing-gradient-gel-electrophoresis (DGGE) analyses. Furthermore, microalgal lipid produced was assessed, based on its fatty acid profile, to meet key biodiesel standards such as saponification, iodine, and cetane numbers.

Acid-catalyzed hot-water extraction of lipids from Chlorella vulgaris.

Acid-catalyzed hot-water treatment for efficient extraction of lipids from a wet microalga, Chlorella vulgaris, was investigated. For an initial fatty acids content of 381.6mg/g cell, the extracted-lipid yield with no heating and no catalyst was 83.2mg/g cell. Under a 1% H2SO4 concentration heated at 120°C for 60min, however, the lipid-extraction yield was 337.4mg/g cell. The fatty acids content, meanwhile, was 935mg fatty acid/g lipid. According to the severity index formula, 337.5mg/g cell of yield under the 1% H2SO4 concentration heated at 150°C for 8min, and 334.2mg/g cell of yield under the 0.5% H2SO4 concentration heated at 150°C for 16min, were obtained. The lipids extracted by acid-catalyzed hot-water treatment were converted to biodiesel. The biodiesels fatty acid methyl ester (FAME) content after esterification of the microalgal lipids was increased to 79.2% by the addition of excess methanol and sulfuric acid.

Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites

The consumption of energy and resources such as water in the cultivation and harvesting steps should be minimized to reduce the overall cost of biodiesel production from microalgae. Here we present a biocompatible and rapid magnetophoretic harvesting process of oleaginous microalgae by using chitosan-Fe3O4 nanoparticle composites. Over 99% of microalgae was harvested by using the composites and the external magnetic field without changing the pH of culture medium so that it may be reused for microalgal culture without adverse effect on the cell growth. Depending on the working volume (20-500 mL) and the strength of surface magnetic-field (3400-9200 G), the process of harvesting microalgae took only 2-5 min. The method presented here not only utilizes permanent magnets without additional energy for fast harvesting but also recycles the medium effectively for further cultivation of microalgae, looking ahead to a large scale economic microalgae-based biorefinement.

Effect of barium ferrite particle size on detachment efficiency in magnetophoretic harvesting of oleaginous Chlorella sp.

Microalgal biofuel is garnering many positive and promising reviews as a fuel for the next generation while research effort continues to improve the efficiency of its harvesting for commercial success. In this report, magnetophoretic harvesting of microalgae is conducted through a three-step process, which includes functionalization of magnetic particles by (3-aminopropyl)triethoxysilane (APTES), magnetic separation, and detachment of magnetic particles by increasing pH to higher than the isoelectric point. Detachment process is specifically focused and found that the use of larger magnetic particles is more efficient for detachment of magnetic particles from algae-particle conglomerates. The detaching efficiency improves from 12.5% to 85% when the particle size is increased from 108 nm to 1.17 μm. Smaller magnetic particles provide larger contact area to microalgae and form strong electrostatic binding to negatively-charged microalgae when pH is lower than the isoelectric point.

Aminoclay-templated nanoscale zero-valent iron (nZVI) synthesis for efficient harvesting of oleaginous microalga, Chlorella sp. KR-1

Synthesis of aminoclay-templated nanoscale zero-valent iron (nZVI) for efficient harvesting of oleaginous microalgae was demonstrated. According to various aminoclay loadings (0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 aminoclay–nZVI ratios), the stability of nZVI was investigated as a function of sedimentation rate. Aminoclay-coated nZVI (aminoclay–nZVI composites) showed optimal dispersibility at the 1.0 ratio, resulting in the smallest aggregated size and uniform coating of aminoclay nanoparticles onto nZVI due to electrostatic attraction between nZVI and aminoclay nanoparticles. This silica-coated nZVI composite (ratio 1.0) exhibited a highly positively charged surface (∼+40 mV) and a ferromagnetic property (∼30 emu g−1). On the basis of these characteristics, oleaginous Chlorella sp. KR-1 was harvested within 3 min at a > 20 g L−1 loading under a magnetic field. In a scaled-up (24 L) microalga harvesting process using magnetic rods, microalgae were successfully collected by attachment to the magnetic rods or by precipitation. It is believed that this approach, thanks to the recyclability of aminoclay–nZVI composites, can be applied in a continuous harvesting mode.

Repeated use of stable magnetic flocculant for efficient harvest of oleaginous Chlorella sp.

In the present study, a simple magnetic-particle recycling strategy was developed for harvest of the oleaginous microalga Chlorella sp. KR-1. The method entails the flocculation of microalgal cells and bare-Fe3O4 magnetic particles (bMP) by electrostatic attraction and the subsequent recovery of the bMP from the harvested flocs by electrostatic repulsion below and above the isoelectric points (IEP), respectively. For 10 recycles, the bMP showed 94-99% and 90-97% harvest and recovery efficiencies, respectively. Furthermore, neither the use of bMP nor pH adjustment showed any adverse effect on the microalgal cell growth or the co-existing bacterial species, as confirmed from the subsequent medium-recycling test and denaturing gradient gel electrophoresis (DGGE) analysis.

Breaking dormancy: an energy-efficient means of recovering astaxanthin from microalgae

Haematococcus pluvialis, in the dormant aplanospore (cyst) status after 30 d of cultivation, accumulates high levels of a superpotent antioxidant, astaxanthin, which has been demonstrated to have enormous therapeutic benefits. However, owing to the robust structure of its trilayered cell wall, the recovery of astaxanthin from the cyst cells remains an energy-intensive process. In the present study, a novel strategy utilizing a short-period germination based on the natural life cycle of H. pluvialis was developed as an energy-efficient pretreatment for the extraction of astaxanthin using ionic liquids (ILs) as green solvents. The germination resulted in damage and deconstruction of the cyst cell wall, and thereby facilitated the extraction of astaxanthin by ILs at room temperature. By this natural pretreatment with 1-ethyl-3-methylimidazolium ethylsulfate for a very short reaction time of 1 min, a high astaxanthin yield of 19.5 pg per cell was obtained, which was about 82% of a conventional volatile organic solvent extraction by strong, 30 000 psi French-pressure-cell homogenization. The maximal astaxanthin-extraction yield from H. pluvialis cells was observed for 12–18 h germination. The germination rate furthermore could be improved by manipulating the nutritional composition (especially the nitrate concentration) of the culture medium. In light of these results, it can be posited that natural germination following the principles of green chemistry can be a uniquely simple method of robust microalgal cyst cell pretreatment and extraction of astaxanthin with room-temperature ILs.

Downstream integration of microalgae harvesting and cell disruption by means of cationic surfactant-decorated Fe3O4 nanoparticles

Microalgal biofuel, albeit an exciting potential fossil-fuel-replacement candidate, still requires the development of more advanced downstream processing technology for its price competitiveness. The major challenge in a microalgae-based biorefinery is the efficient separation of microalgae from low-concentration culture broth. The post-harvesting cell-disruption step necessary to render microalgae suitable for lipid extraction, moreover, further raises energy consumption and cost. For the mitigation of biorefinery complexity and costs, we suggest herein a new scheme that integrates the critical downstream processes (harvesting and cell disruption) by means of cationic surfactant-decorated Fe3O4 nanoparticles. The cationic surfactants’ quaternary ammonium heads play an important role in not only flocculating negatively charged microalgae but also weakening thick cell walls. In the present study, the harvesting efficiency and cell-damaging effects of three cationic surfactants — cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), and cetylpyridinium bromide (CPB) — were evaluated. The CTAB-decorated Fe3O4 nanoparticles, which were found to be the most effective, achieved a 96.6% microalgae harvesting efficiency at a dosage of 0.46 g particle per g cell. Next, for the purposes of magnetic nanoparticle recycling and high-purity microalgal biomass obtainment, microalgae detachment from microalgae-Fe3O4 flocs was performed by addition of an anionic surfactant, sodium dodecyl sulfate (SDS). The detached CTAB-decorated Fe3O4 nanoparticles showed a steady reuse efficiency of about 80%. Furthermore, microalgae harvesting by CTAB-decorated Fe3O4 nanoparticles could contribute to a great improvement in the total extracted lipid content and greener wet extraction without the additional energy-intensive cell-disruption step, thus demonstrating the cell-disruption ability of CTAB-decorated Fe3O4 nanoparticles.

Alginate microgels created by selective coalescence between core drops paired with an ultrathin shell

We report a highly biocompatible and practical protocol to create alginate microgels for bioactive encapsulation. Double-emulsion drops composed of dual cores enclosed by an ultrathin shell are prepared in a capillary microfluidic device, which exhibit selective coalescence between the cores. When the cores are laden with alginate precursors and divalent ions, respectively, coalescence leads to the formation of alginate microgels in the fused core of double-emulsion drops. The microgel can be rapidly released into a continuous water phase by rupturing the liquid shell. This method neither involves any toxic chemical cues for gelation nor long-term exposure to oil, thereby providing highly biocompatible encapsulation.